Centrifugation affects the purity of liquid biopsy-based tumor biomarkers

Abstract

Biomarkers in the blood of cancer patients include circulating tumor cells (CTCs), tumor-educated platelets (TEPs), tumor-derived extracellular vesicles (tdEVs), EV-associated miRNA (EV-miRNA), and circulating cell-free DNA (ccfDNA). Because the size and density of biomarkers differ, blood is centrifuged to isolate or concentrate the biomarker of interest. Here, we applied a model to estimate the effect of centrifugation on the purity of a biomarker according to published protocols. The model is based on the Stokes equation and was validated using polystyrene beads in buffer and plasma. Next, the model was applied to predict the biomarker behavior during centrifugation. The result was expressed as the recovery of CTCs, TEPs, tdEVs in three size ranges (1-8, 0.2-1, and 0.05-0.2 μm), EV-miRNA, and ccfDNA. Bead recovery was predicted with errors <18%. Most notable cofounders are the 22% contamination of 1-8 μm tdEVs for TEPs and the 8-82% contamination of <1 μm tdEVs for ccfDNA. A Stokes model can predict biomarker behavior in blood. None of the evaluated protocols produces a pure biomarker. Thus, care should be taken in the interpretation of obtained results, as, for example, results from TEPs may originate from co-isolated large tdEVs and ccfDNA may originate from DNA enclosed in <1 μm tdEVs. © 2018 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Keywords: biomarker; centrifugation; circulating cell-free DNA; circulating tumor cells; exosomes; extracellular vesicles; flow cytometry; miRNA; microparticles; tumor-educated platelets.

Figure 1

Centrifugation to isolate biomarker of interest from whole blood. The position of a particle after centrifugation can be described with the stokes equation (Eq. (2)). Centrifugation of particles that are uniformly distributed throughout the sample causes large, high‐density particles to move down to the pellet (below the dashed line), while small, low‐density particles stay in the supernatant (above the dashed line).

Figure 2

Centrifugation of beads diluted in PBS or plasma. The modeled bead recovery in pellet (blue dashed line) and supernatant (black, solid line) compared to measured bead recovery in pellet (open blue diamonds) and supernatant (closed black circles) for three centrifugation conditions.