Whole blood stabilization for the microfluidic isolation and molecular characterization of circulating tumor cells

Abstract

Precise rare-cell technologies require the blood to be processed immediately or be stabilized with fixatives. Such restrictions limit the translation of circulating tumor cell (CTC)-based liquid biopsy assays that provide accurate molecular data in guiding clinical decisions. Here we describe a method to preserve whole blood in its minimally altered state by combining hypothermic preservation with targeted strategies that counter cooling-induced platelet activation. Using this method, whole blood preserved for up to 72 h can be readily processed for microfluidic sorting without compromising CTC yield and viability. The tumor cells retain high-quality intact RNA suitable for single-cell RT-qPCR as well as RNA-Seq, enabling the reliable detection of cancer-specific transcripts including the androgen-receptor splice variant 7 in a cohort of prostate cancer patients with an overall concordance of 92% between fresh and preserved blood. This work will serve as a springboard for the dissemination of diverse blood-based diagnostics.

Liquid Biopsy Preservation Solutions for Standardized Pre-Analytical Workflows—Venous Whole Blood and Plasma

“…Wong et al were able to analyze a panel of 26 genes related to the androgen receptor metabolism as well as EMT and proliferation markers from CTCs using EDTA blood stored for 72 h at 4–8 °C. They used a hematologic cell depletion, microfluidic technology called CTC-iChip [ 95 ]. The possibility of achieving sufficient CTC preservation for up to 96 h in EDTA tubes followed by flow sorting and RNA profiling was studied by Apostolou et al [ 96 •].…”

Liquid biopsies

“…In liquid biopsy, the development of more advanced applications that rely on detailed molecular analysis of CTCs has been limited by the requirement to process fresh blood within few hours of blood draw . This is very important for CTCs analyses, considering the rarity of these cells in blood circulation.…”

“…99 In liquid biopsy, the development of more advanced applications that rely on detailed molecular analysis of CTCs has been limited by the requirement to process fresh blood within few hours of blood draw. 100 is not specifically designed for CTC analysis, as RNA is stabilized after lysis of all cells present in whole blood. 105,106 Especially the reliability of gene expression studies in CTCs is affected by a variety of factors, because not only CTCs but mRNA as well is extremely fragile and sensitive.…”

Single-Cell Analysis of Circulating Tumor Cells: Why Heterogeneity Matters

“…To preserve cell viability, peripheral blood sample should be delivered on ice immediately to the laboratory once collected from a cancer patient. RNA degradation occurs within 2–4 h, and sample processing >5 h after the blood draw may result in >60% loss in CTC yield [128]. Similar to tissue acquisition of solid lesions, these requirements impose practical challenges in hospitals and labs, particularly for longitudinal cohort studies.…”