Vestad Beate, Llorente Alicia, Neurauter Axl, Phuyal Santosh, Kierulf Bente, Kierulf Peter, Skotland Tore, Sandvig Kirsten, Haug Kari Bente F, Øvstebø Reidun
The Blood Cell Research Group, Department of Medical Biochemistry, Oslo University Hospital, Ullevål, Oslo, Norway.
Regional Research Network on Extracellular Vesicles, South-Eastern Norway Regional Health Authority, Norway.
J Extracell Vesicles. 2017 Jul 19;6(1):1344087. doi: 10.1080/20013078.2017.1344087. eCollection 2017.
Current methods for characterisation of extracellular vesicles (EVs) need further standardisation in order to obtain an acceptable level of data comparability. Size and concentration of EVs can be determined by nanoparticle tracking analysis (NTA). However, both the heterogeneity of EVs and the choice of instrument settings may cause an appreciable analytical variation. Intra-assay (within-day, = 6) and inter-assay (day-to-day, = 6) variations (coefficient of variation, % CV) of different preparations of EVs and artificial vesicles or beads were determined using two NanoSight NS500 instruments, located at different laboratories. All analyses were performed by the same operator. The effect of applying identical software settings or instrument-optimised settings for each sample type and instrument was also evaluated. Finally, the impact of different operators and the use of two different software versions were investigated. The intra-assay CVs were 1-12% for both EVs and artificial samples, measured on the same instrument. The overall day-to-day variation was similar for both instruments, ranging from 2% to 25%. However, significantly different results were observed between the two instruments using identical software settings. The effect of applying instrument-optimised settings reduced the mismatch between the instruments, resulting in little to no significant divergences. The impact of using different operators and software versions when analysing silica microspheres and microvesicles from monocytes using instrument-optimised settings on the same instrument did not contribute to significant variation compared to the overall day-to-day variation of one operator. Performance differences between two similar NTA instruments may display significant divergences in size and concentration measurements when analysing EVs, depending on applied instrument settings and technical conditions. The importance of developing a streamlined and standardised execution of analysis, as well as monitoring longitudinal variation parameters on both biological and synthetic samples, should be highlighted.
目前用于表征细胞外囊泡（EVs）的方法需要进一步标准化，以获得可接受水平的数据可比性。EVs的大小和浓度可以通过纳米颗粒跟踪分析（NTA）来确定。然而，EVs的异质性和仪器设置的选择都可能导致明显的分析差异。使用位于不同实验室的两台NanoSight NS500仪器，测定了不同制备的EVs、人工囊泡或珠子的批内变异（日内，n = 6）和批间变异（日间，n = 6）（变异系数，%CV）。所有分析均由同一操作人员进行。还评估了针对每种样本类型和仪器应用相同软件设置或仪器优化设置的效果。最后，研究了不同操作人员以及使用两种不同软件版本的影响。在同一仪器上测量时，EVs和人工样本的批内CV均为1%-12%。两台仪器的总体日间变异相似，范围为2%至25%。然而，在使用相同软件设置时，两台仪器之间观察到了显著不同的结果。应用仪器优化设置的效果减少了仪器之间的差异，导致几乎没有显著差异。在同一仪器上使用仪器优化设置分析来自单核细胞的二氧化硅微球和微囊泡时，不同操作人员和软件版本的影响与一名操作人员的总体日间变异相比，并未导致显著差异。在分析EVs时，两台类似的NTA仪器之间的性能差异可能在大小和浓度测量中显示出显著差异，这取决于应用的仪器设置和技术条件。应强调制定简化和标准化分析流程以及监测生物和合成样本纵向变异参数的重要性。
