Particle size distribution of exosomes and microvesicles determined by transmission electron microscopy, flow cytometry, nanoparticle tracking analysis, and resistive pulse sensing
Particle size distribution of exosomes and microvesicles determined by transmission electron microscopy, flow cytometry, nanoparticle tracking analysis, and resistive pulse sensing
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DOI：
10.1111/jth.12602
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发表时间：
2014-07-01
影响因子：
10.4
通讯作者：
Nieuwland, R.
中科院分区：
文献类型：
--
作者：
van der Pol, E.;Coumans, F. A. W.;Nieuwland, R.
背景：细胞外囊泡计数作为疾病的生物标志物具有临床潜力。在生物样品中，最小和最大的囊泡通常大小相差25倍，浓度相差300 000倍，体积相差20 000倍，散射光相差10 000 000倍。由于这种异质性，目前采用的技术检测浓度范围从10 4至10 12囊泡mL（-1）。目的：研究检测到的囊泡浓度的较大变化是否是由五种广泛使用的技术的最小可检测囊泡尺寸引起的。研究方法：用透射电子显微镜（TEM）、常规流式细胞仪、专用于检测亚微米颗粒的流式细胞仪、纳米颗粒跟踪分析（NTA）和电阻脉冲传感（RPS）测量囊泡和参考珠的尺寸和浓度。结果：对于相同的囊泡样品，每种技术得到不同的尺寸分布和不同的浓度。结论：检测到的囊泡浓度之间的差异主要是由最小可检测囊泡大小之间的差异引起的。NTA的最小可检测囊泡尺寸为70-90 nm，RPS为70-100 nm，专用流式细胞术为150-190 nm，常规流式细胞术为270-600 nm。TEM可以检测到存在的最小囊泡，尽管是在粘附在表面上之后。专用流式细胞术在确定参考珠的大小方面是最准确的，但由于囊泡的折射率的异质性，预计在囊泡上不太准确。然而，专用流式细胞术相对快速，并且允许多重荧光检测，使其最适用于临床研究。
Background: Enumeration of extracellular vesicles has clinical potential as a biomarker for disease. In biological samples, the smallest and largest vesicles typically differ 25-fold in size, 300 000-fold in concentration, 20 000-fold in volume, and 10 000 000-fold in scattered light. Because of this heterogeneity, the currently employed techniques detect concentrations ranging from 10 4 to 10 12 vesicles mL(-1). Objectives: To investigate whether the large variation in the detected concentration of vesicles is caused by the minimum detectable vesicle size of five widely used techniques. Methods: The size and concentration of vesicles and reference beads were measured with transmission electron microscopy (TEM), a conventional flow cytometer, a flow cytometer dedicated to detecting submicrometer particles, nanoparticle tracking analysis (NTA), and resistive pulse sensing (RPS). Results: Each technique gave a different size distribution and a different concentration for the same vesicle sample. Conclusion: Differences between the detected vesicle concentrations are primarily caused by differences between the minimum detectable vesicle sizes. The minimum detectable vesicle sizes were 70-90 nm for NTA, 70-100 nm for RPS, 150-190 nm for dedicated flow cytometry, and 270-600 nm for conventional flow cytometry. TEM could detect the smallest vesicles present, albeit after adhesion on a surface. Dedicated flow cytometry was most accurate in determining the size of reference beads, but is expected to be less accurate on vesicles, owing to heterogeneity of the refractive index of vesicles. Nevertheless, dedicated flow cytometry is relatively fast and allows multiplex fluorescence detection, making it most applicable to clinical research.