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Clinical Focus

Circulating microparticles: challenges and perspectives of flow cytometric assessment Eduard Shantsila1; Silvia Montoro-García1,2; Pilar Gallego1; Gregory Y. H. Lip1 1University

of Birmingham Centre for Cardiovascular Sciences, City Hospital, Birmingham, UK; 2Department of Cardiology, Hospital Universitario Virgen de la Arrixaca, University of Murcia, Spain

Summary Circulating blood microparticles are likely to play a significant role as messengers of biological information. Their accurate quantification and characterisation is challenging and needs to be carefully designed with preferable usage of fresh minimally-processed blood samples. Utilisation of flow cytometers specifically designed for analysis of small-size particles is likely to provide considerable methodological Correspondence to: Prof. Gregory Y. H. Lip University of Birmingham Centre for Cardiovascular Sciences City Hospital, Birmingham B18 7QH, UK Tel: +44 121 507 5080, Fax: +44 121 507 5907 E-mail: [email protected]

advantages and should be the preferable option. This viewpoint manuscript provides a critical summary of the key methodological aspects of microparticle analysis.

Keywords Microparticles, flow cytometry, microvesicles

Received: November 14, 2013 Accepted after minor revision: January 7, 2014 Prepublished online: February 20, 2014 doi:10.1160/TH13-11-0937 Thromb Haemost 2014; 111: 1009–1014

Note: The review process for this viewpoint article was fully handled by Christian Weber, Editor in Chief.

Introduction Complex biological systems are composed of myriads of cells with sophisticated mechanisms of information exchange and delivery of regulatory signals. These systems are largely represented by molecular and cellular levels of signalling, and various circulating cells (e.g. leukocytes and platelets) which would serve as ‘mobile messengers’ for biological signals (1). The gap between the molecular and cellular biological messengers is filled by different subcellular vesicles with potent regulatory properties (2, 3). Their regulatory functions are mediated by both surface receptors to allow interaction(s) with target cells as well as by the internal content of the vesicles, which can fuse with the cell content (3). These vesicles, usually called cellular microparticles (MPs) are constantly present in circulation and can thus be implicated in regulation of remote organs and tissues (4). Cellular MPs are usually defined as heterogeneous vesicles sized 0.1-1.0 μm in diameter, which express surface antigens characteristic of their parent cells (5). The size distinguishes MPs from smaller particles called exosomes, which have diameter of less 0.1 μm, and from larger particles, apoptotic bodies formed by fragments of cells undergoing apoptosis. Initially considered as ‘cell dust’ and by-product of cellular death/apoptosis circulating MPs, at least partly, produced in a regulated manner by living cells as part their biological activity (6). Various cells appear to possess mechanism(s) leading to generation of MPs enriched in specific types of membrane proteins and

sub-membrane molecules (7). This implies the presence of complex signalling pathways that both initiate and execute MP formation. However, the precise biological and clinical roles of MPs are remain poorly understood. This is largely due to existing limitations of available methods for MP quantification and characterisation. In this viewpoint, we aim to summarise the key issues related to the analysis of circulating MPs by flow cytometry, which is the principal tool of their assessment. The viewpoint includes some technical recommendations to keep in mind before designing the flow cytometry protocol for MP characterisation.

Size MPs are defined as membranous vesicles of arbitrary size from 0.1 to 1.0 μm (8). Size is one of the key factors defining these events in flow cytometric analysis, yet one of the main challenges for this technology. This approach has unique observer-independent power of analysis of huge number of cellular events. However, MP size is well below the range of reliable discrimination of conventional flow cytometers (about 5 μm). Whilst this equipment can provide some information on MPs sized 0.5-1.0 μm (as estimated using plastic beads) the smaller MPs are largely left beyond their scope of detection (9). Of note, comparisons of MPs ‘seen’ by atomic microscopy and conventional flow cytometers indicates that small-size MPs (i.e. smaller than 0.5 μm) represent majority of these structures and their exclusion from the analysis may present a major bias (10).

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A commercially available system specifically designed for small particle analysis provides obvious advantages in this respect enabling reliable discrimination of small MPs (▶ Figure 1) (11). Forward scatter properties commonly used as an estimate of cell size is hardly appropriate for event of MP size range and side-scatter provides better MP discrimination (12). Light reflective properties of plastic ‘size’ beads may differ from those of MPs and may not provide a very accurate estimate of MP size (13). Analysis of recent papers (see Suppl. Table 1, available online at www.thrombosisonline.com) shows that older generations of flow cytometers with poor resolution of small event are commonly used for MP analysis. In summary, utilisation of flow cytometers designed to accurately discriminate submicron particles should be used in MP research.

Annexin V binding In contrast to cells, MPs may expose negatively charged phospholipids towards their surface. Living cells employ energy-dependent mechanisms of active shift of negatively charged phospholipids towards the inner side of their surface membranes, a process disrupted by cell death (e.g. apoptosis) (11). The observation that many MPs bind annexin V, which suggests exposure negatively charged phospholipids lead to speculation on apoptotic nature MPs. However, not all MPs bind annexin V and annexin V binding is highly dependent on Ca2+ concentrations (11, 14). Despite this, annexin V is a valuable marker of MPs of different origin, with the advantages of having a relatively high density of the target molecules on MPs and excellent reproducibility of measurements. In summary, annexin V-binding MPs should be analysed under strict control of (Ca2+) concentrations.

A

Sample handling The aim of any analysis of circulating MPs is to obtain information on their levels and characteristics as closely reflective of their in vivo status as possible. Achieving this is only possible with appropriate and timely handling of the blood sample, with particular attention paid to careful blood sample collection and anticoagulant use, sample centrifugation, timing of sample processing (i.e. temporal sample stability), sample freezing, thawing, and storage (15). Anticoagulants based on Ca+ chelation (sodium citrate or EDTA) are commonly used for MP analysis. As mentioned above assessment of annexin V-binding will require a careful replenishment of (Ca2+) concentrations. It is not clear at present whether residual phagocytic activity of monocytes/neutrophils and MP binding by leucocytes occurs in sodium citrate and EDTA-anticoagulated blood and how this can affect MP quantification. Importantly these processes of MP elimination could be counterbalanced by release of new MPs from cells contained in the sample and how this affects the net characteristics of the analysed events. Diverse protocols of blood centrifugation have been utilised for MP analysis (15). The centrifugation aims elimination erythrocytes and other circulating cells to achieve better clarity of the sample run through the flow cytometer with only MPs left to meet a laser beam. However, this approach opens a possibility of destruction of some MPs and fusion of others, and temporal stability of MPs and their phenotype ex vivo remains unclear (16-20). Centrifugation of platelet-rich plasma has been shown to remove 58-93% of MPs with some MP subsets being selectively lost (21-23). The impact of ultracentrifugation on MP recovery from platelet free plasma varies according to different reports (21-23). Indeed, centrifugation could also potentially lead to the artificial ‘generation’ of new microvesicles from some blood cells. Analysis

B

Figure 1: Quantification of apoptotic annexin V+ microparticles. A) Overall microparticle gating based on their size (side scatter); B) Gating of apoptotic annexin V+ microparticles. AnV, annexin V; MP, microparticles.

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Shantsila et al. Circulating microparticles – a viewpoint

of MPs from fresh whole blood samples is achievable and helps avoiding several procedure steps interfering with the MP count (24). Also, it is common to freeze MP-containing samples prior to their batched analysis. Freezing and prolonged storage affects MP quantity, with different types of MPs being preferentially increased or lost after the sample thawing, depending on storage conditions and/or analytical protocols used (11, 18, 20-21, 25). The duration of MP storage also matters with short-term and long-term freezing of the same samples reported to change MP levels in opposite directions (21). Nevertheless, MP quantification agreement from samples under different storage conditions tends to be relatively good and a review of the recent publications shows that MPs are most commonly analysed from frozen samples, with various storage duration and temperature (22). In summary, an ideal protocol of flow cytometric analysis of circulating MPs should be based on analysis of fresh whole blood sample stabilised with appropriate anticoagulant/preservative, which still needs to be established. Centrifugation and freezing of blood samples for MP analysis should be avoided.

MP quantification

Analysis of MP origin and surface epitopes

Different types of MPs

The small size of MPs poses another challenge for their flow cytometric assessment, a small surface area with relative small number of protein molecules for antibody binding. Indeed, a leukocyte with diameter of 10 μm has 100 times bigger surface than a 1 μm MP and 10,000 times bigger than 0.1 μm MPs. The surface of the smallest 0.1 μm is just about 0.12 μm2 with only half of it available for illumination by the flow cytometry laser. Consequently the number of specific molecules available for binding by antibodyflourochrome reagents is relatively small to produce bright signal. Amplification of the signal using a two stage biotin-streptavidin based staining has been successfully used to improve discrimination of MPs of specific origin (e.g. from platelets, monocytes, endothelial cells) (11). MPs tend to show a relatively high degree of non-specific binding. The problem is likely to stem from MP contamination by particles-remnants from necrotic and apoptotic cells, which cannot be eliminated from analysis. Unfortunately, the majority of published papers do not show any figures of representative flow cytometry based gating and acquisition strategies, thus preventing appreciation strengths and weaknesses of many individual study protocols. In summary, epitopes with the highest density on the parental cells should be used to define origin of MPs (e.g. CD42b for platelet-derived MPs, CD14 for monocyte-derived MPs, etc). Multiparametric analyses should be avoided unless absolutely essential. Use of signal-amplification approach should be considered. Nonspecific binding of antibodies should always be considered and evaluated before data acquisition. Representative figures of flow cytometrtc gating and acquisition strategies should be included in the published manuscripts.

Employment of high resolution flow cytometry (e.g. Apogee System) has revealed a uniform present of more than one cluster of small size events in blood samples (▶ Figure 1). The nature of these different clusters is not clear at present and may represent different types of MPs, aggregates of large proteins or other biological phenomena. Conventional flow cytometers may have suboptimal resolution for discrimination of different types of submicron particles. In summary, care should be taken to include into analysis clearly selected types of small blood particles.

Accurate MP quantification is essential for assessment of their pathophysiological and prognostic significance. Utilisation of dualplatform approach of MP quantification based on simultaneous counting of leucocytes (or their subsets), estimation of MP proportion to the number of these leukocytes, which absolute numbers are obtained from haematological analysis is not feasible for MPs due to profound difference in their size. Commercial count beads can be used, but they usually have a size which is substantially higher that the largest MPs, which will require appropriate adjustment of the flow cytometric plot scales to accommodate the count beads. This approach appears to be most commonly used in recently published studies. A volumetric approach aiming direct quantification of MPs in accurately measure sample volumes appears to be an optimal solution. In summary, a direct volumetric method is preferable for MP quantification. Use of ‘count beads’ is an alternative option if the volumetric method is not available. Quantification of MPs can be affected by dilution of the samples.

Quo vadis? Whilst this Viewpoint manuscript provides a rather critical overview of microparticle analysis, supporting information from our review of published data on methodological approaches for flow cytometric analysis of MPs is summarised in Suppl. Table 1 (available online at www.thrombosis-online.com) (26-118). It is clear that the published literature thus far contains considerable heterogeneity in methodology and in some cases, inadequate information (for example, on gating strategy, figures, assay variability, etc). Things would need to improve, and in this Viewpoint articles, we provide some suggestions on the way forward. Indeed, circulating blood MPs are likely to play significant role as messengers of biological information. We strongly believe that their accurate quantification and characterisation is challenging and needs to be carefully designed with preferable usage of fresh minimally-processed blood samples. Utilisation of flow cytometers specifically designed for analysis of small-size particles is likely to

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Key messages 1. An ideal protocol of flow cytometric analysis of circulating MPs should be based on analysis of fresh whole blood sample stabilised with appropriate anticoagulant/preservative, which still needs to be established. 2. Centrifugation and freezing of blood samples for MP analysis should be avoided. In summary, epitopes with the highest density on the parental cells should be used to define origin of MPs (e.g. CD42b for platelet-derived MPs, CD14 for monocyte-derived MPs, etc). 3. Multiparametric analyses should be avoided unless absolutely essential, use of signal-amplification approach should be considered and non-specific binding of antibodies should always be considered and evaluated before data acquisition.

provide considerable methodological advantages and should be the preferable option. Conflicts of interest

None declared.

This viewpoint article reflects the view of its author(s) and is not representative of the view of the Editorial Board or the Publishers.

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