- Top of page
- Discussion and conclusion
- Disclosure of Conflict of Interests
Microparticles and exosomes are cell-derived vesicles present in body fluids that contribute to coagulation, inflammation, cellular homeostasis and survival, intercellular communication, and transport of waste materials [1–3]. The size, concentration, biochemical composition and cellular origin of these biological vesicles contain clinically relevant information [4–6]. However, because of the small size of vesicles (30 nm to 1 μm), they are below the detection range of many currently used techniques . Throughout this article, we will use ‘vesicles’ as a generic term for all types of extracellular, biological vesicle.
Approximately 75% of laboratories apply flow cytometry to detect vesicles in clinical samples . A flow cytometer guides cells and vesicles through a laser beam in a hydrodynamically focused fluid stream. One detector is placed in line with the laser beam, and measures the forward-scattered light (FSC). Other detectors measure the side-scattered light (SSC) and fluorescence intensity perpendicular to the beam. Light scattering by vesicles is essential, as scattering is generally used as the trigger signal telling the instrument that a vesicle is present.
A difficulty with flow cytometry is that the relationship between the measured light scattering and the diameter of vesicles is unknown, resulting in recent discussions about the standardization of vesicle detection [9–11] and in unexplained contradictions between the expected and observed measurement results. For example, according to the literature, the smallest polystyrene beads that can be detected by commercial flow cytometers typically have a diameter between 200 nm and 300 nm [9,12,13]. Because vesicles have a lower refractive index than polystyrene beads, they scatter light approximately 10-fold less efficiently than polystyrene beads . Consequently, the smallest single vesicles that are detectable by flow cytometry must be larger than ∼ 500 nm. Using transmission electron microscopy (TEM) image analysis, we have recently shown that urinary vesicles are smaller than 500 nm , so we would not expect these vesicles to be detected by flow cytometry. Nevertheless, urinary vesicles are detectable by flow cytometry , although, from reference measurements with nanoparticle tracking analysis and atomic force microscopy, we now know that flow cytometry underestimates the concentration of vesicles by ∼ 1000-fold [15,16].
To resolve the contradictions in vesicle detection by flow cytometry, we will first present measurements on polystyrene beads and silica beads of known diameter, concentration, and refractive index. Combined with Mie calculations of the optical scattering power, this allows calibration of the flow cytometer, i.e. relating the detected scattering power to the diameter of single polystyrene or silica beads. On that basis, and using an estimated refractive index, we assess the diameter range of the smallest detectable single vesicles. In addition, we show that 89-nm silica beads, which have optical properties resembling those of vesicles, can be detected by regular flow cytometry, albeit at a higher concentration than used to detect single beads. Using dilution series of silica beads and cell-free urine, we elucidate the underlying mechanisms of vesicle detection.
Discussion and conclusion
- Top of page
- Discussion and conclusion
- Disclosure of Conflict of Interests
We have developed a model that relates the measured light scattering power to the diameter of single vesicles by combining measurements on polystyrene beads and silica beads with Mie calculations. Two mechanisms for vesicle detection by flow cytometry can be derived from the results. First, a single vesicle is counted if its diameter is larger than 300–700 nm, i.e. the smallest detectable single vesicle diameter estimated for our flow cytometer. Second, a swarm of multiple smaller vesicles is counted as a single event signal if the power of light scattered by all vesicles that are simultaneously present in the laser beam exceeds the detection limit. For polydisperse samples, such as vesicles in plasma and urine, counts are generated by a combination of single particle and swarm detection.
If large and small particles are defined as particles scattering more or less light than the detection limit of the flow cytometer, single particle detection is caused by large particles only. As every large particle is counted (Table 1), the concentration as determined by the flow cytometer equals the prepared concentration, and a linear relationship between count rate and prepared concentration is obtained, as demonstrated with the use of large 610-nm silica beads only (Fig. 6C).
Swarm detection, on the other hand, is caused by the detection of small particles only. As multiple small particles have to be simultaneously present in the laser beam to generate a single event signal, the flow cytometer-determined concentration underestimates the real concentration, and the relationship between count rate and prepared concentration is non-linear. Although the diameters of the smallest detectable single polystyrene and silica beads are 203 nm and 204 nm, respectively, we demonstrated that we could detect high concentrations of 89-nm silica beads (Fig. 6A) and vesicles filtered through a 220-nm filter (Fig. 3C,D), owing to swarm detection. For both samples, the flow cytometer-determined concentration is more than 1000-fold lower than the real concentration (Fig. 6C,D), and the relationship between count rate and concentration is non-linear. As the detection limit of the flow cytometer is 1.7 × 10−6 mW and the mean SSC powers of an 89-nm silica bead and a urinary vesicle are 2.2 × 10−8 mW and 1.1 × 10−8 mW, respectively, at least 77 silica beads or 155 vesicles have to be simultaneously present in the laser beam to generate a signal. For both samples, this requirement was fulfilled, as the estimated volume of the laser beam is 54 pL and the concentrations of silica beads and vesicles are 1010 mL−1 and 1.6 × 1010 mL−1, respectively, so that, on average, 540 silica beads or 864 vesicles were simultaneously present in the beam. Figure 7 shows the dilution curves of 89-nm silica beads for high, medium and low flow rates. Within the acquisition range (gray area), the established concentration underestimates the prepared concentration, and the relationship between count rate and prepared concentration is non-linear. As a lower flow rate yields a smaller cross-sectional area of the sample stream and thus a smaller effective beam volume , a higher concentration of 89-nm silica beads is required to generate an event signal. Consequently, the dilution curves in Fig. 7 are shifted to the right with decreasing flow rates.
Figure 7. Flow rate dependency of swarm detection. Frequency or count rate vs. total concentration (logarithmic scales) of 89-nm silica beads at high (63 μL min−1; squares), medium (37 μL min−1; circles) and low (12 μL min−1; triangles) flow rates. The solid lines are linear fits through the data points below the maximum acquisition range. The right vertical axis shows the concentration as determined by the flow cytometer. Data points within the acquisition range (gray area) should be considered reliable. The error bars overlap with the symbols and are omitted to improve legibility. SSC, side-scattered light.
Download figure to PowerPoint
For samples containing a mixture of large and small particles, such as vesicles in plasma and urine, counts are generated by both single particle and swarm detection. If the concentration of small particles is lower than the threshold for generation of a signal, the count rate is dominated by large particles, and a linear relationship between count rate and concentration is obtained (Fig. 6C). However, if the concentration of small particles equals or exceeds the threshold for generation of a signal, the contribution of small and large particles to the count rate depends on the relative size and concentration of particles (Fig. 6C). For cell-free urine, we have shown that small vesicles contribute to the count rate by using a 220-nm filter. Without filtration, a fairly linear relationship between the count rate and the concentration is obtained, indicating that large vesicles, i.e. vesicles larger than 300–700 nm, are present. Vesicles larger than 295 nm were not observed by TEM imaging (Fig. 2), probably because the amount of imaged vesicles did not represent the full population, and because the diameter was affected by preanalytic factors, such as centrifugation, staining, fixation, and adhesion . Note that the concentration as determined by the flow cytometer is 1000-fold lower than the concentration estimated with resistive pulse sensing, which was observed when flow cytometry was compared with other, novel detection methods [15,16]. The presence of multiple vesicles in the laser beam explains why the concentration is underestimated 1000-fold, but, more importantly, swarm detection allows the detection of smaller vesicles than previously thought possible.
Sensitivity increases with collection angle
Although it is often thought that FSC should be used to determine the size of vesicles [8,9,11,30,31], Fig. 4A–D shows that, for our flow cytometer, the SSC detector is more sensitive and has a higher capability to resolve the size of beads than the FSC detector, as confirmed by other groups using instruments with a similar optical layout [28,30,32]. In this section, we will explain this phenomenon. Figure 8 shows the optical detection geometry of a flow cytometer (A) and how this affects the detection of light scattered by a cell (B), a microparticle (C), or an exosome (D). Each cell, microparticle or exosome is illuminated by a laser beam with a constant intensity, which we estimate to be 1.4 × 107 W m−2. The FSC detector is a photodiode that detects light which is scattered under an angle of approximately 0.5–7°, depending on the setup of the instrument. To prevent the laser directly illuminating the FSC detector, both the laser beam itself and the light scattered under an angle smaller than 0.5° are blocked by the so-called obscuration bar, as indicated by the interruption in the red line. The SSC detector is a photomultiplier tube, which is not only more sensitive than the photodiode of the FSC detector, but also detects scattered light over a much broader angle, i.e. 47–133°. The dashed blue lines inside the gray circular diagrams show how much light is scattered in each direction. Figure 8B shows that a cell with a diameter of 5 μm scatters light predominantly in the forward direction, which is in the direction of the FSC detector. Hence, FSC is associated with cell size. Figure 8C shows that a microparticle with a diameter of 500 nm scatters light differently than a cell, i.e. mainly under an angle of 0–10°. In comparison with the cell, the fraction of light scattered in the direction of the FSC detector has decreased, whereas the fraction of light scattered in the direction of the SSC detector has increased. Figure 8D shows that an exosome with a diameter of 50 nm scatters light nearly isotropically. As a result, more light is scattered in the direction of the SSC detector than in the direction of the FSC detector. As organelles are of a similar size as microparticles and exosomes, an increased SSC is commonly associated with the complex anatomy of cells. In fact, the SSC detector is optimized to detect scattering from multiple particles smaller than the wavelength. Thus, to increase the sensitivity for vesicle detection, it is beneficial to select the detector with the largest collection angle. For most regular flow cytometers, this implies using the SSC detector.
Figure 8. Optical detection geometry of the FACSCalibur and the angular intensity distribution of light scattered by a single cell, a microparticle, or an exosome. (A) A particle (black dot) is illuminated by a laser beam (blue solid arrow). Forward-scattered light (FSC) and side-scatterered light (SSC) are collected between 0.5° and 7° (red lines) and between 47° and 133° (green line), respectively. The gray circular diagrams show the angular intensity distribution (blue dashed line) of perpendicularly polarized light scattered by a cell (yellow circle) with diameter 5 μm (B), a microparticle (white circle) with diameter 500 nm, and an exosome (black dot) with diameter 50 nm (C). The scattering angle becomes wider for decreasing cell or vesicle diameters. The bar plots at the bottom show the power (logarithmic scale) scattered in all directions (blue), the power scattered in the FSC direction (red), and the power scattered in the SSC direction (green). The calculation parameters are listed in Table 2.
Download figure to PowerPoint
Implications and limitations
Our model explains several recent observations regarding vesicle detection with flow cytometry. In an effort to standardize vesicle detection, the ISTH Scientific Standardization Committee proposed a protocol that used 500-nm and 900-nm polystyrene beads from Megamix to define a vesicle size gate . They observed that flow cytometer-determined concentrations of vesicles appeared to be consistent among instruments measuring FSC with a relatively wide solid angle (1–19°; Beckman-Coulter), but appeared to be inconsistent among instruments measuring FSC with a low solid angle (0.7–10°; Becton-Dickinson). The low collection angle makes the detection limit strongly dependent on the width of the obscuration bar, which is specific for each individual instrument. As vesicle detection typically takes place near the detection limit, the results differed among the Becton-Dickinson instruments.
Chandler et al. applied the Megamix gating strategy to detect platelet microparticles (PMPs) on the Apogee A40, which has an FSC collection angle of 1–70°, and showed that mainly platelets, which have an average diameter of 2000–5000 nm , were counted . Unlike Chandler et al., Mullier et al. and Robert et al. [10,11] could perfectly distinguish PMPs from platelets by using the same gating strategy on their flow cytometers, which all had an FSC collection angle below 19°. To explain this discrepancy and to show that our calibration procedure is generally applicable, we have determined the calibration factors for the FSC detectors of the Apogee A40 and the Beckman-Coulter FC500 by using their data [9,11]. Figure 9A,B shows the diameters of single vesicles as selected by the Megamix gating strategy for the Apogee A40 and FC500, respectively. The range of diameters of single vesicles gated on the Apogee A40 is 200 nm larger than the range gated on the FC500. Owing to the wider collection angle of the Apogee A40 than of the FC500, the Apogee A40 is more sensitive to the difference in refractive index between polystyrene beads and vesicles than the FC500. Nevertheless, single PMPs are not expected to appear in the gated range for either the Apogee A40 or the FC500, as the measured PMP diameter is far below 500 nm [15,16,32]. Rather, it is the presence of multiple PMPs in the laser beam that makes them detectable by flow cytometry. From reference measurements with novel methods, we know that the concentration of PMPs exceeds 1010 mL−1 , which ensures that multiple vesicles are illuminated simultaneously and is sufficient to generate a single event signal.
Figure 9. Selected vesicle diameter range obtained by applying the Megamix gating strategy on the Apogee A40 vs. the FC500. (A) Measured (symbols) and calculated (lines) forward-scattered light (FSC) power (logarithmic scale) vs. diameter for polystyrene beads (black) and vesicles (circle), for the Apogee A40. The measured data points are adopted from Chandler et al. . The obtained calibration factor is 2.67 × 10−6. The Megamix gating strategy would select single vesicles with a diameter between 1000 nm and 2700 nm. (B) Measured and calculated FSC power vs. diameter for polystyrene beads and vesicles for the FC500. The measured data points are adopted from Robert et al. . The obtained calibration factor is 5.18 × 10−4. The Megamix gating strategy would select single vesicles with a diameter between 800 nm and 2400 nm. The calculation parameters are listed in Table 2.
Download figure to PowerPoint
For functional research on vesicles, the presence of multiple vesicles in the beam may have major consequences. For example, different fluorescence signals corresponding to a ‘single event signal’ may originate from multiple vesicles, each containing a different antibody, which may explain the colocalization of granulocyte (CD66e) and platelet (CD61) markers on tissue factor-exposing vesicles . On the other hand, as all vesicles contribute to the signal, our findings explain why flow cytometry results often correlate with disease.
Our work may lead to a better understanding of vesicle detection by flow cytometry, increased sensitivity by optimizing the instrument-specific settings, and improvements in the standardization of measurements between laboratories, which involves at least four steps. First, the scattering power of beads should be measured for the detector with the largest collection angle and highest sensitivity. Second, the scattering power of beads should be calculated specifically for the used detector by Mie theory. Third, the calibration factor should be determined to quantify the detection signal. Fourth, a gate should be selected on the basis of the quantified optical power. For improved standardization, the relationship between the detected scattering power from multiple vesicles, the volume of the laser beam and the flow rate requires further investigation. For improved vesicle detection, we suggest modification of the hardware of the flow cytometer , and study of the refractive index of vesicles and the medium. In addition, as many studies also use fluorescence to identify vesicles, we suggest performing a detailed analysis on fluorescent beads, although many non-trivial practical and theoretical problems will have to be solved before a comparable study based on fluorescence can be performed . In spite of its limitations, flow cytometry will still have to be the present method of choice, because we do not yet have validated methods for quantitative enumeration and establishment of the cellular source of vesicles.
In conclusion, we have established a model that relates the detected scattering power to the diameter of single vesicles. The gating strategy proposed by the Scientific Standardization Committee collaborative workshop selects single vesicles and cells with diameters between 800 nm and 2400 nm when applied on the FSC detector of regular flow cytometers. However, vesicle detection by regular flow cytometry is relies on two different mechanisms: (i) detection of single, relatively large, vesicles that scatter more light than the detection limit; and (ii) swarm detection – that is, multiple relatively small vesicles are simultaneously illuminated by the laser beam and counted as a single event signal. Swarm detection allows the detection of smaller vesicles than previously thought possible. It explains the finding that flow cytometry underestimates the concentration of vesicles, and it clarifies several observations published by the ISTH Scientific Standardization Committee collaborative workshop on vesicle detection.