Stéphane Robert

Standardization of platelet‐derived microparticle enumeration by flow cytometry with calibrated beads: results of the International Society on Thrombosis and Haemostasis SSC Collaborative workshop

Microparticles (MPs) are sub-micrometer sized vesicles released from cell membranes in response to activation or apoptosis [1]. MPs originating from several cell sources have been described in human plasma. Among them, plateletderivedMPs (PMPs) are believed to account for themajority of circulating MPs in healthy subjects [2]. Their levels are increased in several prothrombotic and inflammatory disorders [1]. In these clinical settings, PMP counts may be useful for identifying patients at risk for vascular disorders and for monitoring response to treatment [3]. However, their clinical use is not fully established, because standardized methodologies for PMP counting are lacking. A previous International Society on Thrombosis and Haemostasis (ISTH) Vascular Biology Subcommittee survey indicated that approximately 75% of laboratories use flow cytometry (FCM) to enumerate MPs in clinical samples. However, a wide variety of preanalytic variables and analytic variables have been reported in the literature, resulting in a wide range of PMP values in platelet-free plasma (PFP) of healthy subjects (100–4000 PMPs lL). This lack of consensus stresses the need for standardization [4]. Three ISTH Scientific and Standardization Subcommittees (SSC Vascular Biology, DIC, and Haemostasis &Malignancy) have initiated a project aimed at standardizing the enumeration of cellularMPs by FCM.Afirstcollaborative workshopwas set up, to: first, establish the resolution and the level of background noise of the flow cytometers currently used in laboratories with respect to the strategy requirements; and second, define the interinstrument reproducibility of PMP enumeration in human plasma. This strategy was based on the use of fluorescent calibrated sub-micrometer beads (Megamix beads; BioCytex, Marseille, France), which allow the window of MP analysis to be reproducibly set [5]. The study included 40 laboratories accounting for 59 flow cytometers, and was performed in two stages (Fig. S1): in stage A, participating laboratories received Megamix beads and were asked to set up the FCM protocol and to validate an instrument protocol adapted from a previously described method [5]. On the basis of forward scatter (FS)/FS channeling (FSC) resolution and background characteristics, stage A results led to acceptance or rejection of the tested instruments, with some time being allowed for technical intervention in order to improve any deficient performance. In stage B, selected laboratories received PFP samples prepared as frozen aliquots by the core laboratory, and were asked to analyze them with the previously validated instrument(s), common reagents, and the FCM protocol established in stage A. A detailed description of the methodology is available in the Supporting Information (Data S1). The purpose of this initial phase was to check whether the instrument to be used to enumerate PMPs demonstrated the required performance with a blend of fluorescent beads with well-known sizes and relative amounts. The instruments were validated on the basis of their capacity to discriminate between 0.5-lm and 0.9-lm Megamix beads using the FS/FSC parameter, as well as their background noise. Instruments detecting < 0.1% of fluorescent bead events among total events were rejected, because such a level of background may impede the electronics functions and induce amajor loss of events owing to coincidences and electronic aborts (Fig. S2A,B). Analysis of the results demonstrated that instruments were heterogeneous with respect to FS/FSC resolution and background noise. Furthermore, the level of performance could vary over time (Fig. S2C). Some of the parameters affecting FS/FSC resolution were identified with Megamix beads. Among them, FS/ FSC gain, FS/FSCmode, neutral density (intensity scavenging) Correspondence: Francoise Dignat-George, UMR-S608, 27 Bd Jean Moulin, 13005 Marseille, France. Tel.: +33 491 385600; fax: +33 491 385602. E-mail: francoise.dignat-george@univmed.fr Journal of Thrombosis and Haemostasis, 8: 2571–2574 DOI: 10.1111/j.1538-7836.2010.04047.x

Impact of pre‐analytical parameters on the measurement of circulating microparticles: towards standardization of protocol

Summary.  Background:  Microparticles (MP) are small vesicles of 0.1–1 μm, released in response to activation or apoptosis. Over the past decade, they received an increasing interest both as biomarkers and biovectors in coagulation, inflammation and cancer. Clinical studies were conducted to assess their contribution to the identification of patients at cardiovascular risk. However, among the limitation of such studies, pre‐analytical steps remains an important source of variability and artifacts in MP analysis.

Levels of circulating endothelial progenitor cells are related to uremic toxins and vascular injury in hemodialysis patients

Summary.  Background: Patients suffering from chronic kidney diseases (CKD) exhibit cardiovascular diseases and profound endothelial dysfunction. CKD patients have reduced numbers of endothelial progenitor cells, but little is known about the factors influencing these numbers. Objectives: Among these factors, we hypothesized that uremic toxins and vascular injury affect endothelial progenitor cells. Patients/methods: Thirty‐eight hemodialysis patients were investigated and compared with 21 healthy controls. CD34+CD133+ immature progenitors, CD34+KDR+ endothelial progenitors cells (EPC) and myeloid EPC (mEPC) were counted in peripheral blood. Levels of uremic toxins β2‐microglobulin, indole‐3 acetic acid, indoxylsulfate, p‐cresylsulfate and homocysteine were measured. Vascular injury was assessed in hemodialysis (HD) patients by measuring aortic pulse wave velocity and plasma levels of endothelial microparticles. In vitro experiments were performed to study the effect of uremic toxins on apoptosis of progenitor cells. Results and conclusions: CD34+CD133+ immature progenitor cell number was negatively correlated with the levels of uremic toxins β2‐microglobulin and indole‐3 acetic acid. In vitro, indole‐3 acetic acid induced apoptosis of CD133+ cells. These data indicate uremic toxins have a deleterious role on progenitor cells, early in the differentiation process. Moreover, mEPC number was positively correlated with markers of vascular injury–pulse wave velocity and endothelial microparticle levels. This suggests that vascular lesions could stimulate progenitor cell mobilization, even in a context of reduced EPC induced by CKD. In conclusion, uremic toxins and vascular injury appear to affect endothelial progenitor cell biology in CKD.

Leukocyte- and endothelial-derived microparticles: a circulating source for fibrinolysis

Background We recently assigned a new fibrinolytic function to cell-derived microparticles in vitro. In this study we explored the relevance of this novel property of microparticles to the in vivo situation. Design and Methods Circulating microparticles were isolated from the plasma of patients with thrombotic thrombocytopenic purpura or cardiovascular disease and from healthy subjects. Microparticles were also obtained from purified human blood cell subpopulations. The plasminogen activators on microparticles were identified by flow cytometry and enzyme-linked immunosorbent assays; their capacity to generate plasmin was quantified with a chromogenic assay and their fibrinolytic activity was determined by zymography. Results Circulating microparticles isolated from patients generate a range of plasmin activity at their surface. This property was related to a variable content of urokinase-type plasminogen activator and/or tissue plasminogen activator. Using distinct microparticle subpopulations, we demonstrated that plasmin is generated on endothelial and leukocyte microparticles, but not on microparticles of platelet or erythrocyte origin. Leukocyte-derived microparticles bear urokinase-type plasminogen activator and its receptor whereas endothelial microparticles carry tissue plasminogen activator and tissue plasminogen activator/inhibitor complexes. Conclusions Endothelial and leukocyte microparticles, bearing respectively tissue plasminogen activator or urokinase-type plasminogen activator, support a part of the fibrinolytic activity in the circulation which is modulated in pathological settings. Awareness of this blood-borne fibrinolytic activity conveyed by microparticles provides a more comprehensive view of the role of microparticles in the hemostatic equilibrium.

Accelerated senescence of cord blood endothelial progenitor cells in premature neonates is driven by SIRT1 decreased expression.

Epidemiological and experimental studies indicate that early vascular dysfunction occurs in low-birth-weight subjects, especially preterm (PT) infants. We recently reported impaired angiogenic activity of endothelial colony-forming cells (ECFCs) in this condition. We hypothesized that ECFC dysfunction in PT might result from premature senescence and investigated the underlying mechanisms. Compared with ECFCs from term neonates (n = 18), ECFCs isolated from PT (n = 29) display an accelerated senescence sustained by growth arrest and increased senescence-associated β-galactosidase activity. Increased p16(INK4a) expression, in the absence of telomere shortening, indicates that premature PT-ECFC aging results from stress-induced senescence. SIRT1 level, a nicotinamide adenine dinucleotide-dependent deacetylase with anti-aging activities, is dramatically decreased in PT-ECFCs and correlated with gestational age. SIRT1 deficiency is subsequent to epigenetic silencing of its promoter. Transient SIRT1 overexpression or chemical induction by resveratrol treatment reverses senescence phenotype, and rescues in vitro PT-ECFC angiogenic defect in a SIRT1-dependent manner. SIRT1 overexpression also restores PT-ECFC capacity for neovessel formation in vivo. We thus demonstrate that decreased expression of SIRT1 drives accelerated senescence of PT-ECFCs, and acts as a critical determinant of the PT-ECFC angiogenic defect. These findings lay new grounds for understanding the increased cardiovascular risk in individuals born prematurely and open perspectives for therapeutic strategy.

More on: calibration for the measurement of microparticles: value of calibrated polystyrene beads for flow cytometry‐based sizing of biological microparticles

See also Chandler WL, Yeung W, Tait JF. A new microparticle size calibration standard for use in measuring smaller microparticles using a new flow cytometer. J Thromb Haemost 2011; 9: 1216–24; Mullier F, Bailly N, Chatelain C, Dogné JM, Chatelain B. More on: calibration for the measurement of microparticles: needs, interests, and limitations of calibrated polystyrene beads for flow cytometry‐based quantification of biological microparticles. This issue, pp 1679–81; Chandler WL, Yeung W, Tait JF. More on: calibration for the measurement of microparticles: the authors respond. This issue, pp 1681–2.

Standardized counting of circulating platelet microparticles using currently available flow cytometers and scatter-based triggering: Forward or side scatter?

Clinical determination of MP counts using flow cytometry has not been fully accepted yet due to the lack of standardization protocols. In the past 5 years, we have proposed two versions of a method with reproducible PMP counts in plasma samples. Both methods use forward scatter (FSC)–based threshold set with reference beads of appropriate sizes; first using 0.5 µm beads and later with 0.3 µm beads. Both systems provide reproducible PMP counts. However, this technique works only with some of currently used commercial flow cytometers. Instruments with limited resolution or generating heterogeneous FSC signals are excluded. Such performances are incompatible with the required interinstrument standardization. Here we show that (i) flow cytometers with sub‐optimal FSC capabilities generally have higher SSC resolution and background rejection capacity, and (ii) that the same biological entities, “dim and bright PMP,” both can be counted using alternative strategies, either as previously described, based on FSC measurements, or as presented here, based on SSC detection. The critical element in the standardization protocol is the use of different sizes of reference beads. This study was designed to permit simultaneous access to both FSC‐ and SSC‐optimized platforms. A new range of about 0.17–0.6 µm eq. (µm‐equivalents) is proposed for an alternative SSC–based MP gate generating the same PMP counts as those obtained in the previously proposed 0.3–1 µm eq. FSC–based MP gate. The two equivalent standardization options reconcile intrinsically different scattering behaviors between SSC‐ and FCS ‐ triggered instruments and open the opportunity for multicenter studies in the future.

Tips and tricks for flow cytometry-based analysis and counting of microparticles

Submicron-sized extra-cellular vesicles generated by budding from the external cell membranes, microparticles (MPs) are important actors in transfusion as well as in other medical specialties. After briefly positioning their role in the characterization of labile blood products, this technically oriented chapter aims to review practical points that need to be considered when trying to use flow cytometry for the analysis, characterization and absolute counting of MP subsets. Subjects of active discussions relative to instrumentation will include the choice of the trigger parameter, possible standardization approaches requiring instrument quality-control, origin and control of non-specific background and of coincidence artifacts, choice of the type of electronic signals, optimal sheath fluid and sample speed. Questions related to reagents will cover target antigens and receptors, multi-color reagents, negative controls, enumeration of MPs and limiting artifacts due to unexpected (micro-) coagulation of plasma samples. Newly detected problems are generating innovative solutions and flow cytometry will continue to remain the technology of choice for the analysis of MPs, in the domain of transfusion as well as in many diverse specialties.

Standardization of microparticle enumeration across different flow cytometry platforms: results of a multicenter collaborative workshop

Essentials The clinical enumeration of microparticles (MPs) is hampered by a lack of standardization. A new strategy to standardize MP counts by flow cytometry was evaluated in a multicenter study. No difference was found between instruments using forward or side scatter as the trigger parameter. This study demonstrated that beads can be used as a standardization tool for MPs.

Platelet and not erythrocyte microparticles are procoagulant in transfused thalassaemia major patients.

The level of circulating platelet‐, erythrocyte‐, leucocyte‐ and endothelial‐derived microparticles detected by high‐sensitivity flow cytometry was investigated in 37 β‐thalassaemia major patients receiving a regular transfusion regimen. The phospholipid procoagulant potential of the circulating microparticles and the microparticle‐dependent tissue factor activity were evaluated. A high level of circulating erythrocyte‐ and platelet‐microparticles was found. In contrast, the number of endothelial microparticles was within the normal range. Platelet microparticles were significantly higher in splenectomized than in non‐splenectomized patients, independent of platelet count (P < 0·001). Multivariate analysis indicated that phospholipid‐dependent procoagulant activity was influenced by both splenectomy (P = 0·001) and platelet microparticle level (P < 0·001). Erythrocyte microparticles were not related to splenectomy, appear to be devoid of proper procoagulant activity and no relationship between their production and haemolysis, dyserythropoiesis or oxidative stress markers could be established. Intra‐microparticle labelling with anti‐HbF antibodies showed that they originate only partially (median of 28%) from thalassaemic erythropoiesis. In conclusion, when β‐thalassaemia major patients are intensively transfused, the procoagulant activity associated with thalassaemic erythrocyte microparticles is probably diluted by transfusions. In contrast, platelet microparticles, being both more elevated and more procoagulant, especially after splenectomy, may contribute to the residual thrombotic risk reported in splenectomized multi‐transfused β‐thalassaemia major patients.