Bachurski Daniel, Schuldner Maximiliane, Nguyen Phuong-Hien, Malz Alexandra, Reiners Katrin S, Grenzi Patricia C, Babatz Felix, Schauss Astrid C, Hansen Hinrich P, Hallek Michael, Pogge von Strandmann Elke
Department I of Internal Medicine, University Hospital of Cologne, Center for Integrated Oncology Cologne-Bonn, CECAD Center of Excellence on ''Cellular Stress Responses in Aging-Associated Diseases'', Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany.
Department I of Internal Medicine, University of Cologne, Cologne, Germany.
J Extracell Vesicles. 2019 Apr 1;8(1):1596016. doi: 10.1080/20013078.2019.1596016. eCollection 2019.
The expanding field of extracellular vesicle (EV) research needs reproducible and accurate methods to characterize single EVs. Nanoparticle Tracking Analysis (NTA) is commonly used to determine EV concentration and diameter. As the EV field is lacking methods to easily confirm and validate NTA data, questioning the reliability of measurements remains highly important. In this regard, a comparison addressing measurement quality between different NTA devices such as Malvern's NanoSight NS300 or Particle Metrix' ZetaView has not yet been conducted. To evaluate the accuracy and repeatability of size and concentration determinations of both devices, we employed comparative methods including transmission electron microscopy (TEM) and single particle interferometric reflectance imaging sensing (SP-IRIS) by ExoView. Multiple test measurements with nanospheres, liposomes and ultracentrifuged EVs from human serum and cell culture supernatant were performed. Additionally, serial dilutions and freeze-thaw cycle-dependent EV decrease were measured to determine the robustness of each system. Strikingly, NanoSight NS300 exhibited a 2.0-2.1-fold overestimation of polystyrene and silica nanosphere concentration. By measuring serial dilutions of EV samples, we demonstrated higher accuracy in concentration determination by ZetaView (% BIAS range: 2.7-8.5) in comparison with NanoSight NS300 (% BIAS range: 32.9-36.8). The concentration measurements by ZetaView were also more precise (% CV range: 0.0-4.7) than measurements by NanoSight NS300 (% CV range: 5.4-10.7). On the contrary, quantitative TEM imaging indicated more accurate EV sizing by NanoSight NS300 (% D range: 79.5-134.3) compared to ZetaView (% D range: 111.8-205.7), while being equally repeatable (NanoSight NS300% CV range: 0.8-6.7; ZetaView: 1.4-7.8). However, both devices failed to report a peak EV diameter below 60 nm compared to TEM and SP-IRIS. Taken together, NTA devices differ strongly in their hardware and software affecting measuring results. ZetaView provided a more accurate and repeatable depiction of EV concentration, whereas NanoSight NS300 supplied size measurements of higher resolution.
细胞外囊泡（EV）研究领域不断拓展，需要可重复且准确的方法来表征单个EV。纳米颗粒跟踪分析（NTA）常用于测定EV浓度和直径。由于EV领域缺乏轻松确认和验证NTA数据的方法，因此质疑测量的可靠性仍然非常重要。在这方面，尚未对不同NTA设备（如马尔文的NanoSight NS300或Particle Metrix的ZetaView）之间的测量质量进行比较。为了评估这两种设备在尺寸和浓度测定方面的准确性和可重复性，我们采用了包括透射电子显微镜（TEM）和ExoView的单颗粒干涉反射成像传感（SP-IRIS）在内的比较方法。对纳米球、脂质体以及来自人血清和细胞培养上清液的超速离心EV进行了多次测试测量。此外，还测量了系列稀释和冻融循环导致的EV减少情况，以确定每个系统的稳健性。令人惊讶的是，NanoSight NS300对聚苯乙烯和二氧化硅纳米球浓度的高估了2.0 - 2.1倍。通过测量EV样品的系列稀释，我们证明与NanoSight NS300（偏差百分比范围：32.9 - 36.8）相比，ZetaView在浓度测定方面具有更高的准确性（偏差百分比范围：2.7 - 8.5）。ZetaView的浓度测量也比NanoSight NS300的测量更精确（变异系数范围：0.0 - 4.7）（NanoSight NS300变异系数范围：5.4 - 10.7）。相反，定量TEM成像表明，与ZetaView（偏差百分比范围：111.8 - 205.7）相比，NanoSight NS300在EV尺寸测量方面更准确（偏差百分比范围：79.5 - 134.3），同时具有相同的可重复性（NanoSight NS300变异系数范围：0.8 - 6.7；ZetaView：1.4 - 7.8）。然而，与TEM和SP-IRIS相比，这两种设备都未能报告低于60 nm的EV直径峰值。综上所述，NTA设备在硬件和软件方面差异很大，会影响测量结果。ZetaView对EV浓度的描绘更准确且可重复，而NanoSight NS300提供了更高分辨率的尺寸测量。
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