Preanalytical Variables for the Genomic Assessment of the Cellular and Acellular Fractions of the Liquid Biopsy in a Cohort of Breast Cancer Patients.

Liquid biopsy allows assessment of multiple analytes, providing temporal information with potential for improving understanding of cancer evolution and clinical management of patients. Although liquid biopsies are intensely investigated for prediction and response monitoring, preanalytic variables are of primary concern for clinical implementation, including categories of collection method and sample storage. Herein, an integrated high-density single-cell assay workflow for morphometric and genomic analysis of the liquid biopsy is used to characterize the effects of preanalytical variation and reproducibility of data from a breast cancer cohort. Following prior work quantifying performance of commonly used blood collection tubes, this study completes the analysis of four time points to assay (24, 48, 72, and 96 hours), demonstrating precision up to 48 hours after collection for assay sensitivity, highly reproducible rare cell enumeration, morphometric characterization, and high efficiency and capacity for single-cell genomic analysis. For the cell-free analysis, both freezing and use of fresh plasma produced similar quality and quantity of cell-free DNA for sequencing. The genomic analysis (copy number variation and single-nucleotide variation) described herein is broadly applicable to liquid biopsy platforms capable of isolating cell-free and cell-based DNA. Morphometric parameters and genomic signatures of individual circulating tumor cells were evaluated in relation to patient clinical response, providing preliminary evidence of clinical validity as a potential biomarker aiding clinical diagnostics or monitoring progression.

A new anion exchange purification method for Cu stable isotopes in blood samples.

The isotopic composition of iron, zinc, copper, and cadmium (δ56Fe, δ66Zn, δ65Cu, and δ114Cd) are novel and promising tools to study the metabolism and homeostasis of trace metals in the human body. Serum δ65Cu has been proposed as a potential tool for diagnosis of cancer in liquid biopsy, and other metals may have similar utility. However, accurate analysis of trace metal isotopes is challenging because of the difficulties in purifying the metals from biological samples. Here we developed a simple and rapid method for sequential purification of Cu, Fe, Zn, and Cd from a single blood plasma sample. By using a combination of 11 M acetic acid and 4 M HCl in the first steps of column chemistry on AG-MP1 resin, we dramatically improve the separation of Cu from matrix elements compared to previous methods which use concentrated HCl alone. Our new method achieves full recovery of Cu, Fe, Zn, and Cd to prevent column-induced isotope fractionation effects, and effectively separates analytes from the matrix in order to reduce polyatomic interferences during isotope analysis. Our methods were verified by the analysis of isotope standards, a whole blood reference material, and a preliminary sample set including five plasma samples from healthy individuals and five plasma samples from cancer patients. This new method simplifies preparation of blood samples for metal isotope analysis, accelerating multi-isotope approaches to medical studies and contributing to our understanding of the cycling of Fe, Zn, Cu, and Cd in the human body. Graphical abstract ᅟ.

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.

CONTEXT: - 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. OBJECTIVE: - 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). DESIGN: - 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. RESULTS: - 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. CONCLUSIONS: - 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.