K.G. Jebsen - Thrombosis Research and Expertise Center (TREC), Department of Clinical Medicine, UiT - The Arctic University of Norway, Tromsø, Norway.
Division of Internal Medicine, University Hospital of North Norway, Tromsø, Norway.
Sci Rep. 2018 Nov 21;8(1):17216. doi: 10.1038/s41598-018-35401-8.
Optimal pre-analytical handling is essential for valid measurements of plasma concentration and size distribution of extracellular vesicles (EVs). We investigated the impact of plasma preparation, various anticoagulants (Citrate, EDTA, CTAD, Heparin), and fasting status on concentration and size distribution of EVs measured by Nanoparticle Tracking Analysis (NTA). Blood was drawn from 10 healthy volunteers to investigate the impact of plasma preparation and anticoagulants, and from 40 individuals from a population-based study to investigate the impact of postprandial lipidemia. Plasma concentration of EVs was measured by NTA after isolation by high-speed centrifugation, and size distribution of EVs was determined using NTA and scanning electron microscopy (SEM). Plasma concentrations and size distributions of EVs were essentially similar for the various anticoagulants. Transmission electron microscopy (TEM) confirmed the presence of EVs. TEM and SEM-analyses showed that the EVs retained spherical morphology after high-speed centrifugation. Plasma EVs were not changed in postprandial lipidemia, but the mean sizes of VLDL particles were increased and interfered with EV measurements (explained 66% of the variation in EVs-concentration in the postprandial phase). Optimization of procedures for separating VLDL particles and EVs is therefore needed before NTA-assessment of EVs can be used as biomarkers of disease.
最佳的样本前处理对于测量细胞外囊泡（EVs）的血浆浓度和大小分布至关重要。我们研究了血浆制备、不同抗凝剂（柠檬酸盐、EDTA、CTAD、肝素）和禁食状态对纳米颗粒跟踪分析（NTA）测量的 EVs 浓度和大小分布的影响。从 10 名健康志愿者中抽取血液，以研究血浆制备和抗凝剂的影响，从基于人群的研究中抽取 40 名个体，以研究餐后脂血症的影响。通过高速离心分离后，通过 NTA 测量 EVs 的血浆浓度，通过 NTA 和扫描电子显微镜（SEM）确定 EVs 的大小分布。不同抗凝剂的 EVs 血浆浓度和大小分布基本相似。透射电子显微镜（TEM）证实了 EVs 的存在。TEM 和 SEM 分析表明，高速离心后 EVs 保留了球形形态。在餐后脂血症中，血浆 EVs 没有变化，但 VLDL 颗粒的平均大小增加，并干扰了 EV 测量（解释了餐后阶段 EV 浓度变化的 66%）。因此，在将 NTA 用于评估 EVs 作为疾病生物标志物之前，需要优化分离 VLDL 颗粒和 EVs 的程序。
