A simple flow cytometry method improves the detection of phosphatidylserine-exposing extracellular vesicles

Abstract

Background: Plasma contains cell-derived extracellular vesicles (EVs), which participate in physiopathological processes and have potential applications as disease biomarker. However, the enumeration of EVs faces major problems, due to their sub-micrometer size and to intrinsic limitations in methods of characterization, mainly flow cytometry (FCM).

Objectives: Our objective is to enumerate EVs in plasma, by taking as the prototype the population of phosphatidylserine (PS)-exposing EVs, which constitute one of the major EV populations and are responsible for thrombotic disorders.

Methods: The concentration of PS-exposing EVs in platelet-free plasma (PFP) of healthy subjects was measured by FCM using either light scattering or fluorescence as the trigger and fluorescent Annexin-5 (Anx5) as the specific label. In addition, PS-exposing EVs were enumerated by electron microscopy (EM) after labeling with Anx5 gold nanoparticles and sedimentation on EM grids.

Results: We show that about 50× more Anx5-positive EVs are detected by FCM when detection is triggered on fluorescence as compared with light scattering. By fluorescence triggering, concentrations of 22 000-30 000 Anx5-positive EVs per μL PFP were determined, using two different flow cytometers. The limit of detection of the fluorescence triggering method was estimated at about 1000-2500 Anx5 molecules. Results from EM suggest that EVs down to 100-150 nm diameter are detected by fluorescence triggering.

Conclusion: This study presents a simple method for enumerating EVs. We believe that this method is applicable in a general context and will improve our understanding of the roles of EVs in pathophysiological situations, which will open avenues for the development of EV-based diagnosis assays.

Keywords: blood plasma; cell-derived microparticles; electron microscopy; flow cytometry; phosphatidylserines.

Figure 1

Flow cytometry (FCM) analysis of a platelet-free plasma (PFP) sample by FS triggering (A,B) and FL triggering (C,D), in the absence of Ca²⁺ (A, C) and in the presence of Ca²⁺ (B, D). (A-D). Each of the four panels presents on the left an FS vs. SS color dot plot and on the right an FL1 vs. SS color dot plot. Thresholds are represented by a solid blue line, while fluorescence positivity gates are represented by a dashed blue line. The positions of 500-nm and 1-μm polystyrene particles are indicated in each FS vs. SS plot (Fig. S2). (A,B) FS triggering analysis. The acquisition time was 10 min. Events labeled with Anx5-Fluo are separated into two groups: events of high fluorescence intensity forming a well-defined cluster are colored blue, and all the other Anx5-positive events are colored red. (C,D) FL triggering analysis. The acquisition time was 1 min, which explains why the cluster of erythrocyte ghosts (colored blue) contains less events than in (B).

Figure 2

Influence of Anx5-F* concentration on the concentrations of Anx5-positive extracellular vesicles (EVs) detected by FS and fluorescence (FL) triggering. Curves representing the concentrations of Anx5-positive EVs (expressed per μL of pure platelet-free plasma) detected by FS (dashed lines) and FL triggering (plain lines) at various Anx5-F* concentrations, for two platelet-free plasma (PFP) samples (represented by squares and circles, respectively). The concentrations of Anx5-F* range from 0 to 2.8 nM. Each point represents the mean ± SD of two independent aliquots measured in duplicate.

Figure 3

Influence of platelet-free plasma (PFP) dilution on the detection of Anx5-positive extracellular vesicles (EVs). Curves representing the concentrations of Anx5-positive EVs in diluted PFP samples detected either by FS (dashed lines) or fluorescence (FL) triggering (plain lines) vs. the reciprocal dilution factor, for two PFP samples (represented by squares and circles, respectively). The calculated linear regression lines are presented. R² values higher than 0.96 were obtained for all regression lines. By extrapolating the regression lines to pure PFP (dilution factor 1 x), Anx5-positive EVs concentrations of 35 666 (squares) and 50 239 (circles) are obtained. Each point represents the mean ± SD of two independent aliquots measured in duplicate.

Figure 4

Determination of the number of Anx5-F* molecules needed to detect individual extracellular vesicles (EVs) by fluorescence (FL) triggering. (A-C) FS triggering analysis of a mixture of Quantum™ fluorescein isothiocyanate (FITC)-5 MESF particles made of six particle populations containing, respectively, 0, 1484, 9828, 47 640, 188 438 and 756 101 MESF. (A) The 7.7-μm fluorescent particles form a homogeneous cluster, located in the upper right corner of the FS vs. SS color dot plot. (B) Histogram of fluorescence intensities, showing four resolved peaks (A,B,C,D) corresponding to particles containing 9828 fluorophores and above, while the 1484 MESF particles form a peak (E) overlapping the blank particles (F), which present some auto-fluorescence. (C) Calibration curve correlating the MESF-FITC of the particles to their mean fluorescence intensity, taken from (B). (D) Enlarged view from the low-fluorescence values of the calibration curve (C). The minimal fluorescence intensity of 2 arbitrary units measured by FL triggering is indicated (horizontal arrow), together with its corresponding MESF value, namely 2500 MESF (vertical arrow). (E) FL1 triggering analysis of the Quantum™ FITC-5 MESF particles. Four peaks (A,B,C,D) corresponding to particles containing 9828 MESF and above are detected, while the population of 1484 MESF particles is not detected. The threshold is represented by a solid blue line.

Figure 5

Gallery of Anx5-positive extracellular vesicles (EVs) observed by EM. (A-E) Representative images of Anx5-positive EVs observed on EM grids after sedimentation. Most EVs present a near-circular shape. Very rare gold-nanoparticles (NPs) are present in the background, illustrating the high specificity of Anx5-gold-NP labeling. Some EVs devoid of Anx5-gold-NPs are labeled with arrows, including 50-nm spherical EVs in (B) and a 2-μm tubular EV in (C). The size of the Anx5-positive EVs is indicated on the images (in nm). Considering that a spherical EV, of radius R and area 4πR², transforms upon sedimentation into a flattened circular pancake, of radius R' and area 2πR'², the radius of the flattened pancake is related to the radius of the original sphere by the relationship R  =  R'√2. The size indicated in the images for the circular EVs is the diameter of the equivalent spheres. Scale bars: (A-D) 200 nm; (E) 2 μm. (F) Size histogram of Anx5-positive EVs. About 80% Anx5-positive EVs are comprised between 100 and 400 nm. Histogram determined over 500 images of EVs recorded on three EM grids. Error bars correspond to SD between the three individual datasets [28].

Figure 6

Phenotyping of Anx5-positive extracellular vesicles (EVs) detected by fluorescence (FL) triggering. A platelet-free plasma (PFP) sample was double labeled with Anx5-Cy5 and either anti-mouse-IgG-PE (A), anti-CD41-mAb-PE (B) or anti-CD235a-mAb-PE (C). FL6 fluorescence was used as a trigger, which means that all the detected events are Anx5-positive EVs. Anti-mouse-IgG-PE was used as an isotypic antibody to create a PE positivity gate on the FL6 vs. FL2 color dot plots. Events positive for only Anx5 are colored blue, while events positive for both Anx5 and a PE-conjugated mAb are colored red. On the bottom row, events labeled with Anx5 and/or a PE-conjugated antibody are backgated and displayed on FS vs. SS color dot plots. (C, top row) A small population of erythrocyte ghosts (EG) is observed in the top right corner (colored green). EG are characterized by their high intensity for both Anx5 and anti-CD235a labeling.