ABSTRACT
Microvesicles (MVs) are submicron-size vesicles released from cell membranes in response to activation or apoptosis.1 They are generally defined as 0.1 μm to 1 μm membrane fragments exposing the anionic phospholipid phosphatidylserine (PS) and membrane antigens representative of their cellular origin. MVs originating from several cell sources have been described in human plasma. Among them, platelet-derived MVs (PMVs) are believed to account for the majority of circulating MVs in healthy subjects, followed by erythrocyte-derived MVs (Ery-MVs).2-4 It is now well recognized that MVs behave as vectors of bioactive molecules, playing a role in blood coagulation, inflammation, cell activation, and cancer spreading.5-7 In clinical practice, circulating MVs originating from blood and vascular cells are elevated in a variety of prothrombotic
and inflammatory disorders; cardiovascular diseases; autoimmune, infectious diseases; and cancers.8-10 In these clinical settings, MVs may give information about nonaccessible tissues (tumors, endothelium, placenta, etc.), correlate with disease activity, have a prognostic value to identify patients with thrombotic or vascular risk, and help in treatment monitoring.11-14 Among the different methodologies available to measure MVs in biological samples, flow cytometry (FCM) is the most commonly used technique.15The goal of this chapter is to review the recent progress in terms of standardization, size resolution, and probing to overcome the current limitations of this methodology. 8.1 Advantages and Limitations of FCM
Flow cytometry (FCM) analyzes cells and particles by suspending them in a stream of fluid and passing them by a flow cell interacting with a laser beam. Scattering of light and fluorescence emissions’ detection allow simultaneous multiparametric analysis of the physical and multiple antigen characteristics of up to thousands of particles per second. Thus, microvesicle (MV) measurement by FCM relies mainly on the combination of their scatter light properties and the expression of phosphatidylserine (PS) and specific antigens detected by fluorescence. FCM features give a high potential for MV analysis; indeed, FCM has the major advantage to identify and quantify MV subpopulations, because each particle is interrogated individually. The multiparameter analysis improves the specificity of MV detection. The speed of this technique contrasts with the sensitive but time-consuming microscopy techniques. Absolute counting is also possible in a wide dynamic range using generally counting beads of a known concentration.16 Finally, this technique is available in many research and clinical laboratories. 8.1.2 Limitations
Most FCM limitations are due to the fact that MVs are very small and heterogeneous in size, ranging from 0.1 μm to 1 μm according to electron microscopy. Currently available flow cytometers (FCMrs)
are unable to count all MVs except the larger ones.17 However, the size of the smallest MV an FCMr can detect is a difficult question to answer. As discussed later in this chapter, sizes of calibration beads are not perfectly equivalent to the size of MVs, meaning that if an FCMr can resolve two small latex beads, this does not imply that it can resolve two MVs of the same size range. In addition, performances of the instruments vary between various types of cytometers and even among individual instruments of the same type and also evolve with time on the same apparatus.18 However, we can estimate that the smallest detected MV is around 400 nm for a standard FCMr (sd-FCMr) and 200 nm for a high-sensitivity FCMr (hs-FCMr) using scatter light parameters. Depending on the antigen density on the MVs and the signal/noise ratio of the probe, events smaller than 200 nm can also sometimes be detected by fluorescence.19 Limitations to count all MVs are technologically driven, being dependent both on the fluorescence sensitivity of the instrument and on the intrinsic resolution limit of instruments for the size-related parameter used for the initial discrimination of interesting events from background noise. It is often quoted that the most commonly used laser wavelength (488 nm) is a major determinant of the limit of detection,20,21 but despite its impact in the physics of light scattering, numerous experiments have proven this statement is not entirely accurate.22,23 Among potential sources of instrument noise that impede MV detection, optical noise has generally the greatest impact over electronic and fluidic noise. Thus, optimizing settings can greatly improve results in MV enumeration.17 Because forward scatter (FS/FSC) has to be measured in a narrow scattering angle range close to a very intense light source (the laser beam itself), more precise alignment is required than for wide-angle scattering or fluorescence measurements.24-26 The position of the obscuration bar, which blocks direct laser light, is also critical. Therefore, fine optical alignment by an experienced technician may be highly beneficial for MV analysis, more than for any other normal FCM application. This optical noise can also come from dust particles and/or optical coupling gel stuck on the side of the flow cell. This noise increases slowly with time. It may be prevented by a protected atmosphere in the cytometer’s lab or reduced by cleaning the external side of the flow cell with optical grade alcohol (e.g., isopropanol) during regular technical service.27 In conclusion, laser alignment and careful optical
cleaning by service technicians are critically needed to improve MV detection by an FCMr. To overcome these limitations, two main strategies have been developed: (a) standardization of the MV window of analysis in a defined but reduced size range in order to try and obtain reproducible results in clinical studies and (b) improvement of the detection of small-size MVs with the introduction of the hs-FCMr.
