Thrombosis Detection and role of circulating in thrombosis

M. J. Mooberry ABSTRACT N.S. Key Microparticles (MPs) are submicron vesicles released from the plasma membrane of eukaryotic cells Department of Medicine, Division of in response to apoptosis or certain activators. MPs have been associated with the processes of coag - Hematology and Oncology, University ulation, inflammation, the immune response, cancer metastasis, and angiogenesis, amongst others. of North Carolina, Chapel Hill, NC, Both phosphatidylserine exposure and the presence of tissue factor in the MP membrane may account USA for their procoagulant nature. Elevated numbers of MPs in plasma have been reported in numerous prothrombotic conditions; however, to date there are few data on true causality linking MPs to the genesis of thrombosis. Numerous methodologies have been employed to characterize and enumerate Hematology Education: MPs, although detection is challenging due to their submicron size. remains the most the education program for the frequently utilized strategy for MP detection; however, it is associated with significant technological annual congress of the European limitations. Pre-analytical and analytical variables can influence the detection of MPs, rendering data Hematology Association interpretation difficult. Additionally, lack of methodologic standardization confounds the issue further, although efforts are currently underway to address this limitation. Together, these issues make com - 2012;6:381-388 parison of study results difficult and remain one of the main obstacles preventing MP analysis from being adopted by clinical laboratories. Moving forward, it will be important for the scientific commu - nity to agree upon standardized procedures for the isolation, detection, characterization, and enumer - Acknowledgements: MJM was ation of MPs. supported by grant NIH grant T32 HL007149, and NSK was supported by RO1 HL095096.

Introduction supports the notion that not all MPs carry PS on their surface 6-10 and that the PS content may Historical perspective vary depending on the of origin and stim - ulus or mechanism by which they are Microparticles (MPs) are thought to have formed. 11 Whether this is due to a true lack of been first described by Chargaff and West 1 in PS exposure or whether the expression is the mid-20 th century as a “precipitable factor” below the detection threshold of conventional present in poor plasma that was techniques is unclear. 12 To complicate matters, endowed with the ability to generate throm - it has also been theorized that the presence of bin. Wolf 2 in 1967 described “platelet dust” a cell-specific antigen on the surface of a MP that was formed as a result of platelet shed - does not necessarily identify its cell of origin. ding. This “platelet dust”, which exhibited Soluble antigens from a different cell type procoagulant activity, was detectable in the may adhere to MPs, or fusion may occur 0.1 to 0.3 mm size range by transmission elec - between MPs from one cell type with the cel - tron micrography after ultracentrifugation of lular membrane of a different cell type, there - diluted plasma. Now understood to be platelet by theoretically setting the stage for release of MPs (PMPs), this observation has led to an a second MP expressing an “adopted” anti - exponential growth in the study of MPs, and gen. 13,14 with it a greater understanding of their biolog - MPs must also be distinguished from two ic relevance. other bioactive vesicles released from cells, namely exosomes and apoptotic bodies (AptB). 15 Exosomes are preformed vesicles Definition less than 100 nm that are generated in endo - cytic multivesicular bodies and released via MPs are defined as heterogeneous, submi - exocytosis upon fusion with the cell mem - cron (0.1 to 1 mm) vesicles released from the brane. They are more homogeneous in size membrane of eukaryotic cells in response to than MPs, carry different membrane antigens, specific stimuli or apoptotic cell death. MPs and play an important role in the immune have an intact phospholipid membrane and response and antigen presentation. 16-18 AptB lack a nucleus but express membrane antigens are produced during the latter stages of cell specific to their cell of origin. 3 The working apoptosis, as their name implies. 19 They are definition of an MP has previously included typically larger than MPs and exosomes (in both the size discrimination, as well as the the range of 1-3 mm), although a few may be presence of phosphatidylserine (PS) on the smaller (0.5 mm). 20 Similar to MPs, they outer membrane. 4,5 Newer evidence, however, express PS on their surface; however, in con -

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trast to MPs, AptB carry DNA and histones, which is one Functions of their hallmarks. 20,21 Once thought to be involved only in coagulation, MPs are now known to be involved in numerous other biologic Sources of circulating MPs functions, including inflammation, modulation of the MPs are most commonly derived from circulating immune response, angiogenesis, vascular function, can - blood cells and endothelial cells, although other cells, cer metastasis/invasiveness, and a growing list of oth - such as tumor cells 22 and smooth muscle cells 23 are capa - ers. 36,37 As a result of the presence of multiple bioactive ble of producing MPs as well. They can be detected in molecules, including ligands, receptors, counter-recep - healthy individuals, and PMPs have generally been tors, mRNA, and microRNA, MPs serve as a storage pool accepted as the most abundant subtype of MP in this set - of effectors that can modulate the biologic properties of ting. 24,25 More recent data suggest that a significant portion other cells. Potential (patho)physiologic mechanisms of PMPs may instead be derived from megakaryocytes include the transfer of membrane receptors, release of and not circulating . 26 In addition, other groups or biolipids, and exchange of genetic information have challenged the dogma that PMPs are the most abun - by transfer of mRNA or microRNA. 28,38 Given the expand - dant subpopulation of circulating MPs in healthy individ - ing list of functions linked to MPs, it is not surprising that uals and have reported that endothelial MPs (EMPs) are the list of disease states in which elevated numbers of more abundant than PMPs. 27 MPs have been reported continues to grow, and now includes multiple inflammatory disorders, cardiovascular Formation and clearance diseases, infectious diseases, such as malaria and HIV, The formation and release of MPs from parent cells and autoimmune disorders. 9,39 typically occurs upon stimulation or induction of apopto - sis. It may be considered a broad primitive response to Microparticles and coagulation stress that is shared by all eukaryotic cells, 28 and is With respect to the studied association(s) with coagula - thought to reflect a dynamic balance between cell prolif - tion processes, the potential hemostatic and/or thrombotic eration, stimulation, and death. 29 Specific stimuli known function of MPs may be related to the presence of PS on to induce formation of MPs include activation by sub - the outer membrane, as well as the possible presence of stances, such as endotoxin or cytokines, partial or com - tissue factor (TF). MP-associated PS provides a catalytic plete lysis, such as by complement, oxidative injury, and surface for the assembly of the enzymatic tenase and pro - high shear stress, 30,31 although this list is not exhaustive. thrombinase complexes that initiate and maintain coagula - Mechanistically, new evidence continues to emerge tion. 40 This function may underlie the contribution of MPs regarding the cellular processes that lead to formation and to both the physiologic process of hemostasis, as well as release of MPs. This topic has recently been extensively the pathologic process of thrombosis. 41 Interestingly, the reviewed, 32 but in brief, loss of cellular membrane phos - surface area of a PMP generated ex vivo has approximately pholipid asymmetry with resultant PS exposure appears 50- to 100-fold higher procoagulant activity than the same to be a critical component of MP formation. This process area on an activated platelet, 42 which may help account for is governed by several phospholipid transporters (“flip - the potential pathogenicity/thrombogenicity of MPs. pase”, “floppase” and “scramblase”), which under basal Tissue factor (TF) is the principal physiological initiator of conditions preserve the normal phospholipid asymmetry coagulation in vivo through its interactions with of the cellular membrane, with PS and phos - FVII/FVIIa and is constitutively expressed by most non - phatidylethanolamine confined primarily to the inner vascular cells. 43 Its presence on monocyte-derived MPs leaflet. Calcium influx also appears to be a necessary pre - and tumor-derived MPs is well established; however, requisite for MP formation, as it contributes to both PS whether PMPs or EMPs truly express TF remains a matter externalization, as well as membrane cytoskeleton of debate. 44,45 Although likely only a small fraction of total remodeling through activation of calpains and caspases TF in the blood, MP-borne TF is likely to be functionally necessary for cleavage of certain cytoskeletal proteins active and may thus contribute to the procoagulant nature such as filamin. Upon stimulation, the loss of phospho - of MPs. Indeed, MPs have been studied in numerous lipid asymmetry along with cytoskeletal disruption even - thrombotic conditions, where elevated numbers have been tually leads to membrane blebbing and MP formation and described, 46 but primarily in cross sectional studies. In the release. Newer evidence points to a potential role for flux remainder of this review, we will examine the challenges of other ions, such as Na +, Cl –, and K –, in PS externaliza - associated with MP detection, and then evaluate the poten - tion, as well as a potential role for mitochondria-associat - tial role of MPs in thrombosis. ed proteins, although further investigation is warranted. Even less is known about the clearance of MPs from the circulation. There is evidence to suggest that the circula - Detection of microparticles tion time of intravascular MPs is very short. 33-35 Others speculate that due to their smaller size and ability to dif - Due to their submicron size, the isolation and detection of fuse more rapidly, MPs can effectively escape phagocyto - MPs can be challenging. Numerous modalities have been sis and therefore survive longer than the activated cells employed to evaluate MPs (Table 1), and although each has from which they originated. 28 Despite these gaps in our its own inherent flaws and limitations, flow cytometry cur - knowledge, it is likely that disruptions in homeostasis rently remains the most frequently utilized methodology. In between MP formation and clearance may occur in patho - addition to the analytical variables related to each approach, logical settings, resulting in an increase in circulating MP pre-analytical variables are also exceedingly important and numbers with resultant deleterious effects, including (but may further confound results across studies, regardless of not limited to) thrombosis. the methodology used. Additionally, the MP field has

| 382 | Hematology Education: the education programme for the annual congress of the European Hematology Association | 2012; 6(1) Amsterdam, The Netherlands, June 14-17, 2012 lacked standardization in methodologies, although efforts Use of a tourniquet, traumatic venipuncture, small-diame - are currently underway to address this issue. Addressing ter needles, and use of vacuum-filled containers may cause these limitations and reaching universally agreed upon hemolysis, platelet activation, or contamination by vascu - standardized methods for the isolation, detection, character - lar cells, any or all of which can falsely increase the num - ization, and enumeration of MPs, which address both the ber of MPs detected. It is therefore standard practice to pre-analytical and analytical variables, will be necessary if discard the first several milliliters of blood to minimize MP assays are to contribute clinically meaningful diagnos - these variables. 11 The type of anticoagulant used for blood tic and/or prognostic information. collection has also been shown to have effects on MP analysis. Specifically, heparin has been shown to cause Pre-analytical variables increased total MP numbers compared with citrate or acid- Numerous pre-analytical variables, most of which per - citrate-dextrose (ACD) in some studies. 27 In a separate tain to the collection and handling of specimens, can study, PMP levels increased over several hours after blood impact the detection and enumeration of MPs. Although collection in citrate compared with baseline levels, with no these variables can theoretically affect results regardless such increase observed in samples anticoagulated in cit - of the detection methodology, the majority of information rate-theophylline-adenosine-dipyridamole (CTAD). 47 addressing pre-analytical variables has been identified in studies utilizing flow cytometry. Major variables include Sample processing the method of blood collection, including type of antico - MP analysis is generally performed on plasma samples. agulant used, timeframe and method for processing sam - There have been concerns that delays in the processing of ples, and method of storage. It is worth briefly reviewing samples could allow time for the ex vivo generation of these factors individually. MPs from blood cells. Probably more importantly, how - ever, is the centrifugation protocol by which plasma is Blood collection obtained. Numerous centrifugation protocols are used Several issues regarding the method of blood collection depending on the laboratory performing the analysis, 48 can contribute to artifactual enhancement in MP numbers. resulting in either platelet poor plasma (PPP) or platelet

Table 1. Alternative methodologies to flow cytometry for the evaluation of microparticles.

Methodology Descriptive information Advantages/disadvantages

Functional assays - Assess the procoagulant activity of MPs Advantages: - MP isolation: capture technique vs. ultracentrifugation - Gives (patho)physiologically relevant data - TF-dependent vs. TF-independent procoagulant activity Disadvantages: - Clot-based vs. chromogenic assay - Numerous “in-house” assays - No agreed upon TF standard - No information on size, quantity, or source

ELISA / Immunoassays - Use of dual antibodies for capture and subsequent labeling Advantages: - Compares detected levels to a standard - High throughput - Allows detection of MP subsets Disadvantages: - May also detect soluble antigens - Semi-quantitative

Atomic force microscopy - Immobilization of MPs on a flat surface through use of a specific antibody Advantages: - Interaction force between cantilever tip and sample depends on their distance, - Ultrasensitive: detection to < 10 nm thus generating topographical information about particle size - Detects MP subsets Disadvantages: - Low throughput - Highly specialized

Impedance-based - Uses the Coulter principle to detect changes in electrical impedance by particles Advantages: flow cytometry in suspension that are proportional to size - Quantitative - Utilizes fluorescently labeled antibodies for antigen detection - Detects MP subsets Disadvantages: - Not widely available

Dynamic light scattering - Detects particles in suspension based upon principles of Brownian motion Advantages: - Particle size calculated using Stokes-Einstein equation - Sensitivity: detection of MPs to 10 nm Disadvantages: - Highly specialized - Performs best with monodisperse particles - Does not detect MP subsets

MP = , TF = tissue factor, FCM = conventional flow cytometry

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free plasma (PFP). Alternatively, high speed centrifuga - practices in hospital laboratories. Additionally, the opti - tion (ultracentrifugation) has also been utilized on PFP by mal centrifugation protocol was determined to be some to pellet the MPs, which are then re-suspended 2,500×g performed twice, with careful attention to avoid before analysis. Depending on the protocol, there is either disruption of the buffy coat after the first spin. the potential for loss of MPs in the sediment or the super - Importantly, no significant increase in MP numbers was natant, as well as the risk of contamination of the sample identified using a single freeze/thaw cycle for up to 12 with platelets resulting in artificial increases in PMP months, although a modest increase in MP counts was counts. When MP analysis is performed immediately, observed. This was likely due to reduced platelet contam - there is probably minimal effect on the results unless the ination. Although these results require validation on a centrifugation speed is high enough to pellet a portion of larger scale, they can serve as a starting point for stan - the MPs with the cells. Higher centrifugation speeds may dardizing the pre-analytical aspects of MP analysis. also cause MPs to aggregate. Conversely, if plasma sam - ples are frozen and analyzed at a later date, the centrifu - Flow cytometry gation protocol is of utmost importance. This is due to the Flow cytometry remains the most commonly utilized fact that any residual platelets may be fractured during the approach to the detection and analysis of MPs. 54 With this freeze/thaw cycle (see below), resulting in an artificial technique, a fluidic system transports a biological sample increase in PS-positive MPs and PMPs. Overall, the goal past a focused laser beam where single particle interroga - is to avoid loss of MPs, thus maintaining sensitivity, with - tion takes place in a flow chamber at a rate of up to thou - out sacrificing specificity through the contamination of sands of particles per second. 55 Scattering of light pro - samples with residual platelets and false increases in MP vides information about size and shape of the particles, numbers. and fluorescence emission gives descriptive information based upon the use of fluorescently labeled antibodies Handling and storage practices against specific cellular antigens. Two different types of As already discussed, methods for storage and handling light scatter are collected, each giving unique informa - of plasma samples can significantly impact the analysis of tion. Forward scatter (FSC) or forward angle light scatter MPs as well. Method of transportation of samples and (FALS) measures light scattered at low solid angles and potential agitation during transport are thought to influ - generally provides information about particle size or ence MP numbers. More widely recognized, however, is cross-section. Side scatter (SSC) or right angle light scat - the impact of storage methods, such as freezing, on MP ter (RALS) is the measurement of light scattered at large detection. Several studies have shown an increase in MP solid angles and generally provides information about numbers when comparing frozen to fresh samples. 49,50 both relative size, as well as internal structure and granu - This has typically been seen with the analysis of PPP and larity. This platform is advantageous in that it provides is generally believed to be due to the fracturing of not only quantitative information in MP analysis but also platelets with resultant artificial increase in PMPs. There qualitative information through immunophenotyping and is, however, the theoretical risk of fracturing MPs, simi - thereby identification of the cellular origin of MPs. larly to platelets, causing an artificial increase in overall Despite its ubiquity, flow cytometry has certain limita - MP numbers even in the absence of contaminating tions in MP analysis, many of which are technologically platelets. 51 Conversely, however, one study demonstrated driven. A recent review 56 addresses these limitations in a decrease in total PS-positive MPs with freezing for any detail, but a brief summary follows. First and foremost, length of time, 27 although this result has yet to be replicat - the ability to identify MPs at the smaller end of the size ed. Other potential factors affecting results include the spectrum is limited with conventional flow cytometry, method of freezing, such as snap-freezing with liquid which has historically been used primarily for cellular nitrogen, as well as the method of thawing, such as slowly analysis (Figure 1). This is due, in part, to the fact that on ice or rapidly at 37°C. Ideally, fresh plasma samples detection of particles with diameters that approach the should be analyzed immediately, minimizing this set of wavelength of light (488 nm) of the laser used for excita - pre-analytical variables. However, in addition to being tion is impaired using FSC. 48 With design modifications in impractical, labor-intensive, and inefficient, this restric - some of the newer generation flow cytometers, this detec - tion would limit the ability for collaborative studies tion capability is approaching 300 nm. 56 Even with this between laboratories. improved detection, however, flow cytometry likely only detects the “tip of the iceberg” when it comes to total MP A step towards pre-analytical variable standardization? numbers. Background noise is another obstacle encoun - Recently, several of the aforementioned pre-analytical tered when detecting submicron particles by flow cytom - variables were evaluated to assess their relative impact on etry. This noise can arise from electronics, fluidics, and/or MP measurement. 52 This was the first study of its kind optics; however, optical noise is generally the most using a flow cytometry enumeration protocol that had impeding. 56 Fluidics noise is due to true contaminating previously been standardized. 53 The three major parame - particles present in the fluid analyzed and can be reduced ters found to impact MP analysis most significantly by using a 0.1 µm filter for both the sheath fluid and all included agitation of samples during transportation, time- buffers, which is recommended for all MP analysis. If delay in processing samples, and the centrifugation proto - profound enough, background noise can “drown out” true col. Both total MP and PMP numbers were found to events thereby rendering them undetectable. increase as early as 1 hour after blood collection; howev - Initial gating for MP analysis is typically based upon er, the authors concluded that the increase in MP numbers size through the use of calibration beads. These plastic during the first 2 hours was moderate in comparison with beads are of a known size, and typically a bead that other factors and thus may be acceptable given current approximates 1 µm is used to set the upper size limit for

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MP detection. All events smaller in size are then interro - influence FSC, including relative refractive indices of gated for PS and/or cellular antigens using fluorescently both particles and suspension medium, presence of sur - labeled antibodies to determine either total MPs or MP face absorptive material, particle shape, and surface subsets. Plastic beads, however, remain an imperfect roughness, 57,58 which are not equivalent between biologi - model for size calibration since factors other than size can cal entities and beads. This has led to some controversy over their appropriate application and use. 59 A proposed solution to this problem is the use of biological entities, such as bacteria or viruses, for size calibration, although this strategy would also need standardization. 60 Another analytical variable that is problematic when using flow cytometry for the analysis of MPs is the use of controls. Most laboratories use isotype controls (ITCs) to set a positive vs. negative discrimination limit when using antibodies for antigenic characterization of MPs. This technique has classically been used for cellular phenotyp - ing, but in recent years, its use even in this field has been challenged. 61-63 Although intended to account for back - ground fluorescence due to nonspecific binding, different ITCs can have various levels of background staining depending on their specificity, concentration, degree of aggregation, and fluorophore:antibody ratio. 64 Furthermore, the degree of nonspecific binding of an ITC may or may not reflect an equivalent degree of nonspecif - ic binding as that of the antibody of interest. Therefore, the use of different ITCs can significantly affect the num - ber of positive events detected, 65 making interpretation of results across studies difficult. Adding to the complexity is the fact that many of the markers, such as for leuko - cyte- and endothelial-derived MPs, are weakly expressed and do not provide a clear separation in fluorescence between positive and negative events, making the use of ITCs even more troublesome. Alternative strategies, such as the use of fluorescence minus one (FMO) controls or antigen negative controls, 65,66 are viable options and should be considered when possible. Additionally, titra - tion of antibodies to ensure the optimal concentration is used is of great importance to help limit the effects of nonspecific antibody binding. 66,67 Due to the numerous analytical variables listed above, an attempt at standardization of PMP enumeration has recently been undertaken. 68 Using a mixture of fluores - cent bead sizes (Megamix TM - Biocytex/Stago) and gating strategies to set both an upper and lower size limit for MP detection, a window of MP analysis could be repro - ducibly set to allow consistent PMP enumeration over time. This reproducibility was demonstrated on both an intra- and inter-instrument basis. Subsequently, the stan - dardization protocol was adopted as part of a workshop organized by the International Society of Thrombosis and Hemostasis (ISTH) in an attempt to validate its use, wherein it was shown to allow the reproducible enumera - tion of PMPs across different flow cytometers and differ - ent centers, though modifications of the protocol were required for certain cytometers. 53 The result of this work - shop will hopefully serve as the first step towards contin - uing efforts at standardizing methodologies for MP analy - Figure 1. Relative size comparison of microparticles (MPs) sis, although other variables, such as use of controls, are and cellular blood elements using flow cytometry. (A) still to be addressed. Representative scatter plot of whole blood by forward scat - ter (FSC) and side scatter (SSC) analysis. Note that the MP gate (lower left) is mostly off-scale. (B) Representative scatter plot of platelet rich plasma (PRP) analysis, with Microparticles and thrombosis lower limit of size detection set at 0.5 µm. Platelet microparticle (PMP) population identified by dual staining for CD41 and Annexin is backgated (blue population). The study of MPs in thrombosis is currently an area of intense research due to their presumed role in the proco -

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agulant response through the actions of PS±TF. While the rospective and case-control studies using different modal - overall goal remains to firmly establish a causative role ities for MP detection, where levels are often found to be for MPs in the pathologic process of thrombosis and elevated in cancer patients, both with and without throm - hence use them as a biomarker of thrombotic risk, to date bosis. 77-82 However, some limited prospective data have there are few data in humans to support true causality. In shown a correlation between elevated MPs at diagnosis contrast, there is an abundance of evidence from in vitro and future occurrence of venous thromboembolism studies and animal models of thrombosis that clearly sup - (VTE), 83 although these findings are limited due to lack of port a potential role of MPs, including TF-positive MPs serial MP measurements over time. One such study that (TF +-MPs), in the mechanisms of thrombus formation. 69-74 prospectively examined serial MP-TF procoagulant activ - A detailed analysis of these data, however, is beyond the ity (MP-TF PCA) in pancreatic cancer patients demon - scope of this brief review. Suffice it to say that there is strated a correlation between increased levels and subse - ample evidence to assert with confidence that there is a quent development of VTE; 84 however, the study conclu - biologically plausible role for MPs in the process of sions were limited due to its small size. To this end, thrombosis in vivo . another prospective trial looking at TF +-MPs numbers Moving from the realm of in vitro studies and animal using impedance based flow cytometry as a predictor for models into the realm of clinical studies analyzing the cancer-associated VTE is currently underway. 85 Caution role of MPs in disease states, the results become mixed should be exercised in comparing these data with the and the evidence uncertain. There are numerous reports in prior study, however, due to the fact that a different the literature of elevated MP numbers, using a variety of methodology (with potential differences in pre-analytical assays, in different prothrombotic conditions (Table 2). variables) will be used. It should be noted, as well, that These data are of limited value, however, due to the retro - other studies have failed to confirm an association spective or case-control design of most of the studies. between elevated MPs and clinical thrombosis in malig - While these studies have undoubtedly shown the exis - nancy. 79,86 Additionally, the limited data linking increased tence of an association between elevated MP numbers and MP-TF activity to VTE have been confined to pancreatic thrombosis, they have yet to establish a causal relation - cancer, which may not be broadly applicable across dif - ship. Additionally, some studies have failed to confirm ferent cancer types. Interestingly, several of the studies the presence of elevated MP numbers in some of the same mentioned above additionally demonstrated that MP-TF prothrombotic conditions. 75,76 Whether this is explained by activity was a strong predictor of overall mortality 77,83 in a true lack of correlation between MP numbers and pancreatic cancer, raising the possibility that MP-TF thrombosis, or more likely, reflects the use of different activity may be a better marker of tumor aggressiveness methodologies ( e.g. , flow cytometry vs. procoagulant and mortality than it is a predictor of VTE risk. This activity assays) or different pre-analytical/analytical vari - hypothesis will need to be borne out in future studies. ables across studies that have used the same approach is The NIH Biomarkers Definitions Working Group has unclear. defined a biomarker as “a characteristic that is objectively An area that has received considerable attention regard - measured and evaluated as an indicator of pathogenic ing a potential association between MPs and thrombosis processes”. 12 With this definition in mind, it is clear that is in patients with underlying malignancy. Here again, the establishment of MPs as a biomarker for thrombosis elevated MP numbers have been reported primarily in ret - will necessitate properly designed prospective trials demonstrating that an increase in MP numbers and/or pro - coagulant activity is predictive of thrombotic risk. With the current lack of standardization in both the pre-analytic Table 2. Prothrombotic conditions with reported increased and analytic variables inherent in MP detection and enu - microparticles. meration, this process will be difficult. It is therefore Prothrombotic condition References imperative that further workshops and exercises aimed at standardization are continued in order to eliminate these Sickle cell disease 87 obstacles.

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