Measurement of Platelet Microparticles

  • Jeffrey I. Zwicker
  • Romaric Lacroix
  • Françoise Dignat-George
  • Barbara C. Furie
  • Bruce Furie
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 788)

Abstract

Platelet microparticles are submicron vesicles that can support thrombin generation on externalized negatively charged phospholipids. Increased numbers of circulating platelet microparticles have been investigated as the basis of hypercoagulability in a variety of prothrombotic conditions. Measurement of platelet microparticles is not standardized and a number of preanalytic considerations can influence accurate analysis. We describe methodology for light scatter-based flow cytometry as well as impedance-based flow cytometry for the enumeration and characterization of platelet microparticles.

Key words

Platelet microparticles Microparticles Impedance-based flow cytometry 

1 Introduction

Originally labeled as “platelet dust,” platelet microparticles promote coagulation activation and thrombin generation following exposure of negatively charged phospholipids (i.e., phosphatidylserine) on the external membrane leaflet (1, 2). The physiologic relevance of platelet microparticles continues to be explored as pathologic increases in numbers of circulating platelet microparticles are implicated in a variety of prothrombotic disorders such as heparin-induced thrombocytopenia (3), sickle cell crisis (4), inflammation (5, 6, 7), and myocardial infarction (8). The plasma concentration of platelet-derived microparticles reported in the literature varies considerably both in healthy individuals and in disease, this is likely due to heterogeneous methodologies for detection as well as considerable variation in preanalytic conditions (9, 10).

Platelet microparticles are generally defined as vesicular structures derived from platelets or megakaryocytes that measure <1 μm in diameter. In contradistinction, platelet exosomes are generally considered smaller (<0.1 μm), more homogenous, and lack phosphatidylserine on the outer membrane leaflet (11). Platelet microparticles may be generated in vitro using both physiologic (e.g., thrombin, collagen, or adenosine diphosphate) (12, 13, 14) or nonphysiologic (e.g., calcium ionophore) (13) agonists. Stimuli for the production of platelet-derived microparticles in vivo may also include shear stress (15, 16), oxidative species (17), complement proteins C5b-9 (18), or CD40 ligand (19). Circulating platelet (CD41+) microparticles were previously assumed to be exclusively derived from the mature platelet following activation. However, murine data suggest that megakaryocytes are the predominant source of CD41+ microparticles under physiologic conditions (20).

Microparticles are commonly characterized by the loss of membrane asymmetry. Phospholipid transporters regulate the asymmetry of membrane phospholipids with phosphatidylserine and phosphatidylethanolamine concentrated on the inner leaflet. Platelet activation is followed by an influx of cytosolic calcium that modulates phospholipid transport activity (i.e., flippase, floppase, scramblase). The exposure of phosphatidylserine on the outer membrane is followed by the formation of platelet microparticles. The relevance of phospholipid transport activity and platelet microparticle generation is exemplified by Scott syndrome, a rare bleeding disorder characterized by deficient scramblase activity, deficient phosphatidylserine exposure, and decreased microparticle generation (21). However, the precise role of phospholipid transport proteins in the generation of platelet microparticles is uncertain as mice deficient in scramblase (PSLCR1 or PLSCR3) do not exhibit loss of phosphatidylserine asymmetry (22, 23), platelet-derived microparticle formation occurs in the absence of phosphatidylserine exposure (24), and a large percentage of circulating platelet microparticles lack externalized phosphatidylserine (25). Other mechanisms that regulate platelet microparticle formation continue to be explored, including apoptotic pathways (i.e., caspase-3) and calpain-mediated phosphorylation of cytoskeletal proteins (26, 27).

1.1 Measurement of Platelet Microparticles by Flow Cytometry

Flow cytometry is the standard technique used to quantify and size platelet-derived microparticles in plasma. It is widely available and provides a reliable means to characterize cells by size and antigen expression thus providing an attractive method to characterize microparticles. However, there are notable limitations in the application of flow cytometry based on light scattering for the analysis of microparticles. Forward scatter is dependent on variables that are independent of size including wavelength of incident light, particle shape, absorptive material, and relative refractive indices of particles and suspension medium (28). One key limitation of the use of light scattering for sizing of biologic microparticles is that polystyrene beads have a much greater refractive index than the microparticles. The latest generation flow cytometers appear to have improved sensitivity for the detection of microparticles although smaller particles are likely below the lower limits of detection (29). Alternative methods for microparticle detection are currently being evaluated, including atomic force microscopy (30), dynamic light scatter (31), capture or activity assays (10, 32), and impedance-based flow cytometry (33).

We currently utilize an impedance-based flow cytometer for microparticle sizing, characterization, and concentration determination (33). This instrument (Cell Lab Quanta SC, Beckman Coulter, Miami, FL) is based on the Coulter principle whereby a particle suspended in an electrolyte solution enters an aperture and displaces an equal volume of electrolyte solute. The displaced solute increases the impedance across the circuit generating a voltage spike that is proportional to the volume of the microparticle. Simultaneous measurement of fluorescence using a 488-nm laser permits characterization of surface antigens. The sensitivity of instrument into the submicron range largely is dependent on the aperture size. Refinement of Coulter-type instruments in the 1970s enabled the characterization of submicron structures including bacteria and viruses (34, 35). The standard diameter aperture for the Cell Lab Quanta SC is 125 μm with a resolution of approximately 2.5 μm. We have modified this instrument using smaller (40 or 25 μm diameter) flow cells to improve submicron resolution. Although this methodology offers an improved sensitivity compared with standard flow cytometers based on light scattering, very small particles cannot be resolved under the current configuration.

2 Materials

2.1 Impedance Analysis

  1. 1.

    Beckman Coulter Cell Lab Quanta SC flow cytometer (Beckman Coulter, Miami, FL) has been refitted with a smaller aperture flow cell. The standard instrument is equipped with a 125-μm diameter flow cell which is not adequate for microparticle analysis. In general, the lower limit of microparticle sizing by impedance is 2% of the aperture diameter. Our current system is configured with a 40-μm diameter aperture for microparticle analysis.

     
  2. 2.

    Fluorescent polystyrene microspheres (Dragon Green 0.78 μm fluorescent microspheres from Bangs Laboratories, Fishers, IN).

     
  3. 3.

    Sheath fluid (Iso-Diluent, Cat#629967, Beckman Coulter, Miami, FL).

     
  4. 4.
    Antibodies.
    1. (a)

      For platelet microparticle analysis we utilize mouse antihuman CD41a-FITC and mouse irrelevant isotype-matched IgG1-FITC (BD Diagnostics, Franklin Lakes, NJ). A number of platelet antigens can be considered for platelet microparticle analysis including antibodies to platelet glycoproteins IIb (CD41), IX (CD42a), Ibα (CD42b), IIIa (CD61), or P selectin (CD62b) (36). The expression of these antigens can be influenced by platelet activation or mechanism of microparticle formation (37, 38). All the concentrations of antibodies must be optimized. The stock concentration of our CD41a-FITC from BD Diagnostics is 6.25 μg/ml while that of isotype-matched control immunoglobulin IgG1-FITC is 50 μg/ml and thus the isotype control antibody is diluted 1:8 in filtered buffer.

       
     

2.2 Standard Flow Cytometry Analysis

  1. 1.

    FC500 Cytomics flow cytometer (Beckman Coulter, Miami, FL) with optimal laser alignment and clean flow cell.

     
  2. 2.

    Megamix Beads (BioCytex, Marseille, France): a blend of monodisperse fluorescent beads with three diameters: 0.5, 0.9, and 3 μm. The specifications of these beads indicate an accurate numerical ratio of 2:1 between the 0.5- and 0.9-μm beads.

     
  3. 3.

    Sheath fluid (Iso-Flow, Beckman Coulter) passed through a 0.22-μm filter.

     
  4. 4.
    Labeling Reagents: For platelet microparticle analysis, we use
    1. (a)

      Annexin V labeled with FITC (Annexin V FITC/7AAD, Beckman Coulter ref PN IM3614).

       
    2. (b)

      Mouse antihuman CD41-PE antibody (clone PL2-49, BioCytex catalogue number 5112-PE100T).

       
    3. (c)

      Mouse antihuman IgG1-PE isotype-matched control antibody (clone 2DNP-2H11/2H12 BioCytex, catalogue number 5108-PE100T)

      All antibodies are centrifuged for 2 min at 13,000  ×  g before use.

       
     
  5. 5.

    Counting beads: Flow count® fluorospheres, Beckman Coulter ref 7547053.

     

3 Methods

A number of strategies have been utilized to evaluate microparticle populations in platelet-free plasma (PFP). Herein is a description of the methodology for impedance-based and light-scatter flow cytometry. However, there are a number of preanalytic ­considerations that can influence platelet microparticle measurements which need to be standardized for accurate analysis (seeNotes 17).

3.1 Enumeration and Sizing of Polystyrene Beads by Impedance-Based Flow Cytometry

  1. 1.
    Prior to running biologic samples, polystyrene beads are evaluated on a daily basis to ensure proper calibration of size and counting (Fig. 1). For these purposes, we utilize fluorescent 0.78-μm beads from Bangs Laboratories. These have a known concentration and are accurately sized by ±0.5%. Based on a stock concentration of 4.2  ×  106/μl, beads are diluted 1:10,000 in filtered sheath solution.
    Fig. 1.

    Calibration of the Cell Lab Quanta SC using polystyrene microspheres. Calibration beads are utilized to validate microparticle measurements. Analysis is performed using 0.78-μm fluorescent beads diluted 1:10,000 in filtered sheath fluid. (a) Histogram of fluorescence events recorded. (b) The same fluorescence events are shown relative to microsphere diameter. The predominance of events are shown at 102 (FL1 arbitrary units) corresponding to a diameter of 0.78 μm. Populations at increased fluorescence and size represent microsphere doublets.

     
  2. 2.

    Aliquot 1.5 ml of vortexed bead dilution into sample cup

     
  3. 3.

    Set fluorescence photomultiplier tube voltage (PMT) such that beads are detected between the 102 and 103 FL1 units on the x-axis. Note that fluorescent beads typically require lower PMT voltage than biologic specimens labeled with fluorescent antibodies. The fluorescent threshold for detection of events is set above 101. Events are triggered based on fluorescence rather than electronic volume.

     
  4. 4.

    Analyze size and concentration of polystyrene beads to ensure accuracy of the instrument.

     

3.2 Measuring Microparticles by Impedance-Based Flow Cytometry

  1. 1.
    Each sample will be analyzed in the presence of irrelevant ­isotype immunoglobulins and again in the presence of antigen-specific antibodies. Antibody concentrations need to be optimized. For platelet microparticle analysis using BD Diagnostics CD41a antibodies as above the antibody (20 μl) is added to 40 μl of plasma sample (Fig. 2).
    Fig. 2.

    Measurement of plasma microparticles by impedance-based flow cytometry. The presence of different populations of microparticles can be determined using fluorescent antibodies in platelet poor plasma with a Cell Lab Quanta SC. (a) Irrelevant isotype-matched antibodies are used to establish background fluorescence PMT voltage. The rectangular region defining microparticles is based on size (diameter <1 μm) and fluorescence (FL1). (b) Antigen-positive microparticles are quantified in the rectangular region above baseline fluorescence (x-axis, diameter; y-axis, fluorescence).

     
  2. 2.

    Incubate 30 min in the dark.

     
  3. 3.

    Perform a 1:50 dilution of sample-antibody solution sheath solution passed through a 0.22-μm filter prior to use in the assay (e.g., 10 μl with 490 μl of sheath solution).

     
  4. 4.

    Analyze the microparticles treated with isotype-matched ­control antibody and set fluorescence PMT voltage such that background fluorescence is just above lower discriminator (101 units on FL1). This is illustrated in Fig. 2, where microparticle events are captured based on a diameter set between 0 and 1.0 μm and fluorescence (FL1) above the background observed for fluorescently labeled isotype-matched antibodies.

     
  5. 5.

    Setup software analysis for two samples (one treated with isotype-matched control antibody and one treated with specific antibody) to be run in triplicate.

     
  6. 6.

    Analyze labeled samples.

     
  7. 7.

    The concentration of platelet microparticles is calculated by subtracting the background number of microparticles determined using the isotype-matched control from the number of microparticles determined using the antigen-specific antibody.

     

3.3 Measuring Microparticles by Light Scattering-Based Flow Cytometry

Light scattering-based flow cytometry is the method most commonly used for analysis of microparticle populations. Forward light scatter is the most appropriate parameter to analyze microparticle size. The limit of resolution on forward scatter between instrument noise and microparticles depends, in part, on fine optical adjustments, fluidics, and clean optics. These variables are prone to change over time and can vary between platforms. Therefore, in order to obtain reliable and reproducible results on standard flow cytometers, a bead-based strategy is recommended to identify the lower sensitivity of size-related forward scatter (39). These latex beads are not intended to give an accurate sizing of the microparticle population but to standardize analysis. It should be noted that because manufacturers of flow cytometers do not use the same optical design for forward scatter measurements results may vary for different instruments (29, 40). However, with adequate optical centering the relative position between the same reference beads and microparticle populations does not appear to vary significantly for specific cytometry instruments.

3.3.1 Standardization Protocol

This protocol establishes methods for reproducible measurement of platelet microparticles using a standardized window with Megamix beads (Fig. 3). Megamix beads are run every day to check the stability of the instrument. The 0.9-μm beads identify the upper limit of the microparticle region; the 0.5-μm beads identify the forward scatter threshold and lower microparticle limit at a standardized level corresponding to its median value. The analysis is performed using the intrinsic 2:1 ratio of 0.5 μm beads to 0.9 μm beads, always including the same percentage of the 0.5-μm beads in the analysis. The following steps describe how to create the standardized window for microparticle analysis.
Fig. 3.

Measurement of plasma microparticles by scatter-based flow cytometry using a standardized protocol. Platelet microparticles can be reproducibly measured on a standardized window by Megamix beads using a Cytomics FC500. Upper panel  : Construction of the microparticle (MP) region in the microparticle protocol. (a) On a FS (forward scatter) log SS (side scatter) log cytogram, the microparticle region is defined by the lower part of the FS cut-off (discriminator, shown here as a dotted line), set up using the 0.5-μm bead percentage, and the upper part of the 0.9-μm bead autogate. (b) FS distribution of the Megamix submicrometer beads. In this bimodal histogram gated on the union of singlets from the 0.5 to 0.9 μm beads, the percentage of 0.5 μm beads (region J) varies with FS settings. The value of 48.7% shown here corresponds to a cut-off that rejects about half of the 0.5-μm beads. Lower panel  : Platelet microparticle (PMP) analysis in the microparticle (MP) protocol. (c) Phosphatidylserine (PS+) PMP forward scatter. PS  +  PMPs are seen as red dots among background (gray dots). (d) Dual fluorescence analysis of a platelet-free plasma stained with annexin V (AnnV)–fluorescein isothiocyanate (FITC) and anti-CD41–phycoerythrin (PE). PS  +  PMPs are represented as red dots, and background noise or other MPs as gray dots (this figure is kindly provided by S. Robert).

Step 1

  1. 1.

    Run Megamix beads with discriminator (threshold) on FL1 parameter.

     
  2. 2.

    Gate each individual bead subset on SS (Side Scatter)  ×  FL1 cytogram.

     
  3. 3.

    Build forward scatter histogram for 0.5 and 0.9 μm beads.

     
  4. 4.

    At this step, all the 0.5 and 0.9 μm beads are included in the analysis; so, percentages of 66 and 33% should be found for 0.5 and 0.9 μm beads, respectively.

     

Step 2

  1. 1.

    Run Megamix beads with discriminator on forward scatter parameter.

     
  2. 2.

    Monitor percentage of 0.5 μm beads (left peak) and optimize forward scatter settings to approach 50%. This threshold level is selected as the lower limit for microparticle analysis.

     
  3. 3.

    Visualize side scatter/forward scatter cytogram. Create an elliptical autogate on the 0.9 μm beads. Set up the microparticle gate to tangent the autogate. This defines the upper limit of the microparticle window.

     

3.4 Measuring Platelet Microparticles by Forward-Based Scatter Flow Cytometry

  1. 1.

    Mix 30 μl of PFP with 10 μl CD41-PE  +  10 μl AnnV-FITC (Tube 3). For the negative control, the same volume of PFP is mixed with 10 μl CD41-PE  +  10 μl AnnV-FITC (Tube 1, which will be diluted in a buffer without calcium) and 10 μl IgG1-PE  +  10 μl AnnV-FITC (Tube 2). Vortex.

     
  2. 2.

    Importantly, antibody concentrations were adapted by BioCytex so that 10 μl CD41-PE matched (12.5 μg/ml) with 10 μl IgG1-PE (25 μg/ml).

     
  3. 3.

    Incubate 30 min at room temperature with samples protected from light.

     
  4. 4.

    Add 500 μl of binding buffer (2.5 mM CaCl2 in HEPES buffer) to tubes 2 and 3 and 500 μl of PBS (phosphate-buffered saline) to tube 1.

     
  5. 5.

    Vortex Flow-Count® fluorospheres for 10 s and add 30 μl to each vial.

     
  6. 6.

    Keep stained samples at room temperature and immediately proceed to flow cytometry analysis.

     
  7. 7.

    Run each labeled sample using the analysis protocol described above (time stop set to 1 min; low flow-rate), with a maximum delay of 30 min after the end of labeling.

     
  8. 8.

    Analyze platelet microparticles in a dual fluorescence cytogram (FL1  ×  FL2) gated on the microparticle scatter gate established above.

     
  9. 9.

    Set FL1 and FL2 compensations using the samples from tubes 1 and 2.

     
  10. 10.

    Platelet microparticles are defined as AnnV+/CD41+  ±  AnnV−/CD41+ events. Absolute values are determined using counting beads (Flow-Count, Beckman-Coulter) as follows: platelet microparticles (events/μl)  =  (platelet microparticle events  × beads concentration)/beads events.

     

This standardized strategy provides reliable results for platelet microparticle enumeration. However due to limitations in resolution, very small platelet microparticles are not detected under these conditions. Recent technological improvements of different cytometry instruments, such as Gallios (Beckman Coulter) and BD influx (Becton Dickinson) have partially overcome these limitations opening the avenue for improved detection of microparticle populations of smaller sizes (29).

3.5 Immunoassays

Enzyme linked immunosorbent assays (ELISA) can also be utilized for the analysis of platelet microparticles (32). These assays have certain potential advantages including increased sensitivity for the evaluation of microparticles below the resolution of flow cytometry or identification of particles with weakly expressed antigens. For instance, 96-well plates can be coated with antiplatelet antibodies (e.g., antiglycoprotein IX) to bind platelet microparticles. The number of platelet microparticles can be quantified using biotinylated antiglycoprotein Ib using a peroxidase substrate (32). Michelsen et al. described a method that quantifies platelet microparticles by an immunofluorometric assay using lanthanide (europium chelate) as time-delayed fluorescent reporter (41). Hybrid assays such as Zymuphen-MP® technology (Hyphen Biomed, Andresy, F) combine solid-phase capture of MP on platelet antibodies and determine prothrombinase activity (42, 43).

4 Notes

Accurate measurement of platelet microparticles in plasma requires the standardization and optimization of a number of preanalytic variables. Manipulations from venipuncture to thawing a specimen can influence the number of platelet microparticles measured in a specimen. The optimal methods to control for such variables have not been established but may include the following:
  1. 1.
    Venipuncture
    • Platelet activation may occur during phlebotomy. Perform venipuncture with a 20- or 21-gauge needle applied to the antecubital vein following the application of a light tourniquet. The first 2–3 ml of blood should be discarded (38, 44).

     
  2. 2.
    Collection tubes
    • Activation of platelets prior to centrifugation can generate platelet microparticles. Sodium citrated Vacutainer® tubes (BD Diagnostics, Franklin Lakes, NJ) are most commonly utilized to prevent platelet activation. The addition of platelet inhibitors as contained in CTAD tubes (0.109 M buffered sodium citrate, 15 mM theophylline, 3.7 mM adenosine, and 0.198 mM dipyridamole) may be more effective in inhibiting in vitro platelet microparticle formation (38, 45, 46, 47). EDTA leads to dissociation of platelet glycoprotein IIb/IIIa heterodimers (48, 49) and platelet activation thus should be avoided (36).

     
  3. 3.
    Transportation
    • Transporting blood in upright rather than horizontal position may limit the number of platelet microparticles formed in vitro (50).

     
  4. 4.
    Time to centrifugation
    • Platelet-derived microparticles increase over time following blood collection (51). Microparticle numbers appear to be stable up to 2 h in citrated or CTAD tubes (45, 47).

     
  5. 5.
    Centrifugation
    • The centrifugation speeds and time vary widely among studies (44). Typically, the initial spin to generate cell-free plasma is 1,500–2,500  ×  g for 15–20 min. However, platelets may persist after a single centrifugation step (36) and an additional centrifugation step of 13,000  ×  g for 2 min ensures PFP (39, 52). Following centrifugation, the plasma should be carefully aspirated leaving the bottom 1 cm undisturbed (9).

     
  6. 6.
    Storage
    • If samples must be stored prior to analysis, snap freezing of PFP at −80°C can be considered (39).

     
  7. 7.
    Thawing
    • Different methods have been described for thawing microparticle samples including slow thaw on ice (9). Thawing for several minutes at 37°C is preferred by some investigators (45, 53, 54). A single freeze–thaw does not increase platelet microparticle numbers appreciably but repeated freeze–thaw cycles may significantly alter the number of platelet microparticles (4, 51, 55). Stability of platelet microparticles should be confirmed for specific applications.

     

Notes

Acknowledgment

We thank S. Robert for his input on forward scatter-based flow cytometry.

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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Jeffrey I. Zwicker
    • 1
  • Romaric Lacroix
    • 2
    • 3
    • 4
  • Françoise Dignat-George
    • 2
    • 3
    • 4
  • Barbara C. Furie
    • 1
  • Bruce Furie
    • 1
  1. 1.Division of Hemostasis and Thrombosis, Beth Israel Deaconess Medical CenterHarvard Medical SchoolBostonUSA
  2. 2.UMR-S608 INSERMMarseilleFrance
  3. 3.Faculté de PharmacieUniversité de la MéditerranéeMarseilleFrance
  4. 4.Service d’hématologie, CHU La Conception, AP-HMMarseilleFrance

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