Medical Cell BioPhysics, University of Twente, Enschede, the Netherlands.
Amsterdam UMC, University of Amsterdam, Laboratory of Experimental Clinical Chemistry, Amsterdam, the Netherlands.
Cytometry A. 2018 Dec;93(12):1207-1212. doi: 10.1002/cyto.a.23641.
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.
癌症患者血液中的生物标志物包括循环肿瘤细胞（CTCs）、肿瘤衍生的血小板（TEPs）、肿瘤衍生的细胞外囊泡（tdEVs）、EV 相关 miRNA（EV-miRNA）和循环无细胞 DNA（ccfDNA）。由于生物标志物的大小和密度不同，需要对血液进行离心以分离或浓缩感兴趣的生物标志物。在这里，我们根据已发表的方案，应用模型来估计离心对生物标志物纯度的影响。该模型基于 Stokes 方程，并使用缓冲液和血浆中的聚苯乙烯珠进行了验证。接下来，该模型被应用于预测生物标志物在离心过程中的行为。结果以 CTCs、TEPs、tdEVs 在三个大小范围内（1-8、0.2-1 和 0.05-0.2μm）、EV-miRNA 和 ccfDNA 的回收率表示。珠子回收率的预测误差<18%。最显著的混杂因素是 1-8μm tdEVs 中 22%的 TEPs 污染和<1μm tdEVs 中 8-82%的 ccfDNA 污染。Stokes 模型可以预测血液中生物标志物的行为。评估的方案中没有一个能产生纯的生物标志物。因此，在解释获得的结果时应谨慎，例如，TEPs 的结果可能源自共同分离的大 tdEVs，而 ccfDNA 可能源自包裹在<1μm tdEVs 中的 DNA。2018 年，作者。细胞仪分析部分由 Wiley 期刊出版公司代表国际细胞分析促进协会出版。
