Amsterdam UMC location University of Amsterdam, Vesicle Observation Centre, Laboratory of Experimental Clinical Chemistry, Department of Clinical Chemistry, Meibergdreef 9, Amsterdam, The Netherlands.
Cancer Centre Amsterdam, Imaging and Biomarkers, Amsterdam, The Netherlands.
J Extracell Vesicles. 2023 Feb;12(2):e12302. doi: 10.1002/jev2.12302.
Human blood plasma prepared by centrifugation contains not only extracellular vesicles (EVs) but also platelets and erythrocyte ghosts (ery-ghosts). Here we studied whether analysis of miRNA associated with plasma EVs (EV-miRNA) is affected by the presence of platelets and ery-ghosts. EDTA blood was collected from healthy donors (n = 3), and plasma was prepared by the centrifugation protocol recommended by the International Society on Thrombosis and Haemostasis (ISTH), and by a centrifugation protocol from an EV-miRNA expert lab (non-ISTH protocol). EVs were isolated from plasma by size-exclusion chromatography CL-2B (SEC2B), and concentrations of platelets, activated platelets, ery-ghosts and EVs (150-1000 nm) were measured by calibrated flow cytometry. Two EV-associated miRNAs (let7a-5p and miR-21-5p), and one platelet-associated miRNA (miR-223-3p), were measured by qRT-PCR. Measurements were performed with and without filtration using 0.8 μm track-etched filters to remove platelets and ery-ghosts from plasma and EV-enriched SEC fractions. Plasma prepared by both centrifugation protocols contained platelets and ery-ghosts, which co-migrated with EVs into the EV-enriched SEC2B fractions. Filtration removed platelets and ery-ghosts (>97%; p ≤ 0.05) and did not affect the EV concentrations (p > 0.17). The miRNA concentrations were 2-4-fold overestimated due to the presence of platelets but not ery-ghosts. Thus, filtration of human plasma is expected to improve comparability and reproducibility of quantitative EV-miRNA studies. Therefore, we recommend to measure and report the plasma concentration of platelets for EV-miRNA studies, and to filter plasma before downstream analyses or storage in biobanks.
人血浆经离心制备后不仅含有细胞外囊泡（EVs），还含有血小板和红细胞（ery-ghosts）。在这里，我们研究了与血浆 EVs 相关的 miRNA（EV-miRNA）分析是否受到血小板和 ery-ghosts 存在的影响。从健康供体（n=3）采集 EDTA 血液，并按照国际血栓和止血学会（ISTH）推荐的离心方案和 EV-miRNA 专家实验室的离心方案（非 ISTH 方案）制备血浆。通过尺寸排阻色谱 CL-2B（SEC2B）从血浆中分离 EVs，并通过校准流式细胞术测量血小板、活化血小板、ery-ghosts 和 EVs（150-1000nm）的浓度。通过 qRT-PCR 测量两种 EV 相关 miRNA（let7a-5p 和 miR-21-5p）和一种血小板相关 miRNA（miR-223-3p）。使用 0.8μm 轨迹蚀刻滤器进行过滤和不进行过滤，以去除血浆和 EV 富集 SEC 级分中的血小板和 ery-ghosts，进行了测量。两种离心方案制备的血浆均含有血小板和 ery-ghosts，它们与 EV 一起迁移到 EV 富集的 SEC2B 级分中。过滤去除了血小板和 ery-ghosts（>97%；p≤0.05），但不影响 EV 浓度（p>0.17）。由于血小板的存在，miRNA 浓度高估了 2-4 倍，但红细胞没有。因此，过滤人血浆有望提高定量 EV-miRNA 研究的可比性和重现性。因此，我们建议在进行下游分析或在生物库中储存之前，对 EV-miRNA 研究进行测量并报告血小板在血浆中的浓度，并对血浆进行过滤。
