Extracellular vesicles (EVs) have sparked tremendous interest owing to their prominent potential in diagnostics and therapeutics. Isolation of EVs from complex biological fluids with high purity is essential to the accurate analysis of EV cargo. Unfortunately, generally used isolation techniques do not offer good separation of EVs from non-EV contaminants. Hence, it is important to have a standardized method to characterise the properties of EV preparations, including size distribution, particle concentration, purity and phenotype. Employing a laboratory-built nano-flow cytometer (nFCM) that enables multiparameter analysis of single EVs as small as 40 nm, here we report a new benchmark to the quality and efficiency assessment of EVs isolated from plasma, one of the most difficult body fluids to work with. The performance of five widely used commercial isolation kits was examined and compared with the traditional differential ultracentrifugation (UC). Two to four orders of magnitude higher particle concentrations were observed for EV preparations from platelet-free plasma (PFP) by kits when compared with the EV preparation by UC, yet the purity was much lower. Meanwhile, the particle size distribution profiles of EV preparations by kits closely resembled those of PFP whereas the EV preparation by UC showed a broader size distribution at relatively large particle size. When these kits were used to isolate EVs from vesicle-depleted PFP (VD-PFP), comparable particle counts were obtained with their corresponding EV preparations from PFP, which confirmed again the isolation of a large quantity of non-vesicular contaminants. As CD9, CD63 and CD81 also exist in the plasma matrix, singleparticle phenotyping of EVs offers distinct advantage in the validation of EVs compared with ensemble-averaged approaches, such as Western blot analysis. nFCM allows us to compare different isolation techniques without prejudice.

Nasopharyngeal carcinoma (NPC) is a malignant tumor commonly associated with Epstein−Barr virus (EBV) infection, and its early diagnosis as well as its differentiation from nasopharyngitis (NPG) remains challenging due to the insufficient sensitivity of routine screening methods in clinical practice. To date, circulating extracellular vesicles (EVs, 40−1000 nm) have shown appealing potential in liquid biopsy for cancer diagnosis and prognosis. Herein, nanoflow cytometry (nFCM) capable of single EV analysis was applied to examine the expression of surface proteins with very low copy numbers on individual EVs as small as 40 nm. The particle concentrations of five EV subsets exposing EBVencoded latent membrane proteins (LMP1 and LMP2A) and tumor markers (PD-L1, EGFR, and EpCAM) in plasma were determined rapidly via single-particle enumeration. We identified a five-marker panel named EV SUM5 (an unweighted sum of the concentration of the five individual EV subsets) that significantly surpassed the traditional VCA-IgA assay in discriminating NPC patients from both healthy donors and NPG patients with accuracies of 96.3 and 83.1%, respectively. Moreover, EV SUM2 (an unweighted sum of virus-specific LMP1-and LMP2A-positive EVs) could achieve the diagnosis of NPG with an accuracy of 82.6%. Collectively, the work presented a rapid, reliable, and noninvasive method as well as two diagnostic markers to help more accurately differentiate NPC from NPG patients and healthy donors in clinical practice.

Although lipophilic membrane dyes (LMDs) or probes (LMPs) are widely used to label extracellular vesicles (EVs) for detection and purification, their labelling performance has not been systematically characterized. Through concurrent side scattering and fluorescence detection of single EVs as small as 40 nm in diameter by a laboratory‐built nano‐flow cytometer (nFCM), present study identified that (1) PKH67 and PKH26 could maximally label ∼60%–80% of EVs isolated from the conditioned cell culture medium (purity of ∼88%) and ∼40%–70% of PFP‐EVs (purity of ∼73%); (2) excessive PKH26 could cause damage to the EV structure; (3) di‐8‐ANEPPS and high concentration of DiI could achieve efficient and uniform labelling of EVs with nearly 100% labelling efficiency for di‐8‐ANEPPS and 70%–100% for DiI; (4) all the four tested LMDs can aggregate and form micelles that exhibit comparable side scatter and fluorescence intensity with those of labelled EVs and thus hardly be differentiate from each other; (5) as the LMD concentration went up, the particle number of self‐aggregates increased while the fluorescence intensity of aggregates remained constant; (6) PKH67 and PKH26 tend to form more aggregated micelles than di‐8‐ANEPPS and DiI, and the effect of LMD self‐aggregation can be negligible at optimal staining conditions. (7) All the four tested LMDs can label almost all the very‐low‐density lipoprotein (VLDL) particles, indicating potential confounding factor in plasma‐EV labelling. Besides, it was discovered that DSPE‐PEG2000‐biotin can only label ∼50% of plasma‐EVs. The number of LMP inserted into the membrane of single EVs was measured for the first time and it was confirmed that membrane labelling by lipophilic dyes did not interfere with the immunophenotyping of EVs. nFCM provides a unique perspective for a better understanding of EV labelling by LMD/LMP.