NIH, National Cancer Institute, Division of Cancer Treatment and Diagnosis (DCTD) NIH - National Institutes of Health National Cancer Institute DCTD - Division of Cancer Treatment and Diagnosis

Centrifugation affects the purity of liquid biopsy-based tumor biomarkers.

Author(s): Rikkert LG, van der Pol E, van Leeuwen TG, Nieuwland R, Coumans FAW

Publication: Cytometry A, 2018, Vol. 93, Page 1207-1212

PubMed ID: 30551256 PubMed Review Paper? No

Purpose of Paper

This paper used a model to predict the effects of centrifugation speed on recovery of circulating tumor cells (CTCs), tumor-educated platelets (TEPs) and tumor-derived extracellular vesicles (tdEVs) of different sizes, EV associated miRNA (EV-miRNA), and circulating cell-free DNA (ccfDNA). Results from the model were confirmed by investigating recovery of beads from plasma.

Conclusion of Paper

The predicted recovery of beads based on particle diameter in plasma with centrifugation at 300 x g or 2700 x g was within 8% of that found experimentally, but the model overestimated particle speed with centrifugation at 15,800 x g, resulting in errors as high as 20%. The model predicted that isolation of ccfDNA from small tdEVs, TEPs from large tdEVs, CTCs from large tdEVs and TEPs, and EV-miRNA from ccfDNA is not possible using centrifugation alone.

Studies

  1. Study Purpose

    This study used a model to predict the effects of centrifugation speed on recovery of CTCs, TEPs and tdEVs of different sizes, EV-miRNA, and ccfDNA and confirmed the model by investigating recovery of beads from plasma. The authors constructed a model based on the Stokes equation using published values for plasma density and viscosity and published values for CTC, tdEV, EV-miRNA, and ccfDNA density and diameters. The model was used to calculate recovery at a variety of centrifugation speeds and rotor diameters. Results from the model predictions were compared to experimental results of bead recovery from plasma using beads of different diameters. Whole blood was drawn into EDTA vacutainers using a 21-gauge needle from healthy volunteers. Plasma was obtained by double centrifugation at 2,500 x g for 15 min at 20˚C within 20 min of blood collection. The model was tested by the addition of beads of different diameters (400-3005 nm) prior to plasma centrifugation at 300 x g for 20 min, 2700 x g for 22 min, and 15800 x g for 60 min. Bead recovery was then measured by side-scatter flow cytometry

    Summary of Findings:

    The predicted recovery of beads based on particle diameter in plasma with centrifugation at 300 x g or 2700 x g was within 8% of that found experimentally, but the model overestimated particle speed with centrifugation at 15800 x g, resulting in errors as high as 20%. The model predicted that centrifugation at 800 x g for 10 min would result in 100% recovery of CTCs but that the plasma would also contain large tdEVs, tEPs, and ccfDNA. The model predicted that further purification using EpCAM beads would eliminate the TEPs and ccfDNA but that CTCs would still contain some tdEVs. Model predictions indicated that the TEP protocol (centrifugation at 120 x g for 20 min followed by 360 x g for 20 min) would allow for recovery of 71% of TEPs and 22% of large tDEVs, with <3% of CTCs, small tdEVs, and ccfDNA retained. The model predicted the EV miRNA centrifugation protocol (900 x g for 7 min, 2500 x g for 10 min and 500 x g for 10 min) would allow for recovery of 40% of intermediate tdEVs, 57% of small tdEVs, and <1% of large tdEVs, but also 57% of ccfDNA; however, the authors state the ccfDNA could be removed by size exclusion chromatography. The model showed the Speicher ccfDNA protocol (200 x g for 10 min followed by 1600 x g for 10 min twice) would allow for recovery of 82% of ccfDNA but also 82% of small tdEVs and 63.4% of intermediate tdEVs. In contrast, the Dawson ccfDNA protocol (820 x g for 10 min followed by 20000 x g for 10 min) recovered only 48% of ccfDNA but contamination was also decreased as only 6% of intermediate tdEVs and 49% of small tdEVS were retained.

    Biospecimens
    Preservative Types
    • None (Fresh)
    Diagnoses:
    • Normal
    Platform:
    AnalyteTechnology Platform
    Cell count/volume Flow cytometry
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Biospecimen Aliquots and Components Centrifugation Different number of centrifugation steps compared
    Multiple speeds compared
    Multiple durations compared

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