Cellular microvesicles and nanovesicles (exosomes) are involved in many disease processes and have major potential as biomarkers. However, developments in this area are constrained by limitations in the technology available for their measurement. Here we report on the use of fluorescence nanoparticle tracking analysis (NTA) to rapidly size and phenotype cellular vesicles. In this system vesicles are visualized by light scattering using a light microscope. A video is taken, and the NTA software tracks the brownian motion of individual vesicles and calculates their size and total concentration. Using human placental vesicles and plasma, we have demonstrated that NTA can measure cellular vesicles as small as ∼50 nm and is far more sensitive than conventional flow cytometry (lower limit ∼300 nm). By combining NTA with fluorescence measurement we have demonstrated that vesicles can be labeled with specific antibody-conjugated quantum dots, allowing their phenotype to be determined.From the Clinical EditorThe authors of this study utilized fluorescence nanoparticle tracking analysis (NTA) to rapidly size and phenotype cellular vesicles, demonstrating that NTA is far more sensitive than conventional flow cytometry.

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Extracellular vesicles (EVs) are membrane-bound complexes secreted from cells under both physiological and pathological conditions. They contain proteins, nucleic acids and lipids and act as messengers for cell–cell communication and signalling, particularly between immune cells. EV research is a rapidly evolving and expanding field, and it appears that all biological fluids contain very large numbers of EVs; they are produced from all cells that have been studied to date, and are known to have roles in several reproductive processes. This review analyses the evidence for the role of EVs throughout human reproduction, starting with the paternal and maternal gametes, followed by the establishment and continuation of successful pregnancies, with specific focus, where possible, on the interaction of EVs with the maternal immune system. Importantly, variations within the EV populations are identified in various reproductive disorders, such as pre-term labour and pre-eclampsia.

BackgroundThe placental syncytiotrophoblast releases micro and nanovesicles (STBM), into the maternal circulation in normal pregnancy and in increased amounts in pre-eclampsia (PE), which have proinflammatory and antiangiogenic activity and are implicated in PE pathophysiology. Better characterisation of STBM is essential to understand their role in PE.Methods and ResultsSTBM prepared by placental lobe dual perfusion (pSTBM) and mechanical disruption (mSTBM) were analysed by four colour flow cytometry (4CFC), nanoparticle tracking analysis (NTA) and Western blotting to determine vesicle size, purity and Flt-1 and endoglin (Eng) expression. Biological activity of STBM associated Flt-1 and endoglin was assessed by the ability of VEGF, PlGF and TGFβ to bind to mSTBM and inhibit mSTBM induced endothelial monolayer disruption. STBM content was consistently high (∼87–95%) across the different preparations. However, surface antigen intensities differed, with significantly lower placental alkaline phosphatase (P<0.05) and Eng (P<0.05) expression on mSTBM, and Flt-1 (P<0.05) expression on pSTBM. For PE placenta derived preparations, pSTBM contained lower Eng positive STBM (P<0.05) and mSTBM Eng expression was increased (P<0.05). Western blotting revealed increased Flt-1/sFlt-1 (P<0.02) and decreased placental alkaline phosphatase (P = 0.0002) content of PE placenta pSTBM. Using NTA, perfused PE placentas released significantly larger MV (P<0.001). Finally, VEGF, PlGF and TGFβ bound to mSTBM at physiologically relevant concentrations and inhibited mSTBM induced endothelial disruption (P<0.05-P<0.001).ConclusionsThis study has found differences in physical and antigenic characteristics of normal and PE placenta STBM preparations produced by placental perfusion or mechanical disruption. We have also demonstrated that large quantities of biologically active STBM associated endoglin and Flt-1/sFlt-1 could contribute to the increased circulating levels measured in PE patients and add to the perturbation of the maternal vascular endothelium, normally attributed to non-membrane bound sFlt-1 and sEndoglin.

The release of extracellular vesicles (EV) by the syncytiotrophoblast (STB) may be an important mechanism by which the placenta signals to the mother. STB derived EV (STBEV) are comprised predominantly of exosomes (50-150nm) and microvesicles (100-1000nm) that contain bioactive mediators such as proteins, nucleic acids and lipids. They, along with larger syncytial nuclear aggregates are released by the STB into the maternal circulation throughout gestation in normal pregnancy where they appear to have an immunoregulatory role, inhibiting T cell and NK cell responses. In pre-eclampsia (PE) STBEV are released in significantly increased numbers and have pro-inflammatory, anti-angiogenic and procoagulant activity, implicating them in the maternal systemic inflammation, endothelial dysfunction and activation of the clotting system which typifies the disorder. Research has focused on understanding the biological significance of STBEV by measuring their size and repertoire of molecules carried and how they differ in normal pregnancy and PE, using techniques such as Nanoparticle Tracking Analysis, flow cytometry and mass spectrometry. We have also found alterations in STBEV surface glycans associated with PE. The goal is to better understand the role STBEV play in normal pregnancy and PE and whether they are potential biomarkers of placental pathology and therapeutic targets in PE.

IntroductionOptical techniques are routinely used to size and count extracellular vesicles (EV). For comparison of data from different methods and laboratories, suitable calibrators are essential. A suitable calibrator must have a refractive index (RI) as close to that of EV as possible but the RI of EV is currently unknown. To measure EV, RI requires accurate knowledge of size and light scattering. These are difficult to measure as most EVs cannot be resolved by light microscopy and their diameter is smaller than the wavelength of visible light. However, nanoparticle tracking analysis (NTA) provides both size and relative light scattering intensity (rLSI) values. We therefore sought to determine whether it was possible to use NTA to measure the RI of individual EVs.MethodsNTA was used to measure the rLSI and size of polystyrene and silica microspheres of known size and RI (1.470 and 1.633, respectively) and of EV isolated from a wide range of cells. We developed software, based on Mie scattering code, to calculate particle RI from the rLSI data. This modelled theoretical scattering intensities for polystyrene and silica microspheres of known size (100 and 200 nm) and RI. The model was verified using data from the polystyrene and silica microspheres. Size and rLSI data for each vesicle were processed by the software to generate RI values.ResultsThe following modal RI measurements were obtained: fresh urinary EV 1.374, lyophilised urinary EV 1.367, neuroblastoma EV 1.393, blood EV 1.398, EV from activated platelets 1.390, small placental EV 1.364–1.375 and 1.398–1.414 for large placental EV (>200 nm). Large placental EV had a significantly higher RI than small placental EV (p<0.0001). The spread of RI values was narrower for small EV than for the more heterogeneous large EV.DiscussionUsing NTA and Mie scattering theory, we have demonstrated that it is possible to estimate the RI of sub-micron EV using NTA data. EV typically had a modal RI of 1.37–1.39, whereas values of >1.40 were observed for some large (>200 nm) microvesicles.ConclusionThis method for measuring EV RI will be useful for developing appropriate calibrators for EV measurement.