Vascular Biology
DRAFT Subcommittee Minutes
23 July 2011
9:00-13:00
Room E
Chairman: Françoise Dignat George (FR)
Co-chairmen: Michael Berndt (IE), John Griffin (US), Nigel Key (US), Peter Newman (US), Florence Toti (FR)
This year, the SSC VB meeting was focused on microparticles .The session was divided into two sections: the Educational session, devoted to microparticle function, generation and role in disease, and the business session, focused on Microparticle determination and standardization, This business session was divided into three parts addressing respectively: Part A, "Standardized strategies". Part B "Functional assays", and Part C "Novel methodologies." The success of this SSC VB was attested by the presence of at least 200 participants during the whole session
The Educational session, chaired by Michael Berndt and Nigel Key, covered the relevance of microparticles (MP) as clinical biomarkers of vascular disease, the multifaceted role of endothelial derived MP and the role of MP in cancer.
Guus Sturk (NLD) presented an overview of the definition of different vesicle subtypes including exosomes, microparticles and apoptotic bodies. Particular attention was paid to the methodological recommendations related to their isolation from different biological fluids but also to the most appropriate markers and methods used for phenotypic characterization and enumeration. He highlighted their involvement in various biological functions and also reviewed the pathological circonstances associated with altered MP levels. The role of MP as potential prognostic markers to identify patients with vascular risk but also as a therapeutic monitoring strategy to evaluate the effect of medications impacting the cardiovascular system, was underlined
Francoise Dignat George (France) presented the multiple faces of endothelial derived MP (EMP), that can be viewed as a “miniature version” of endothelial cells, expressing a large repertoire of antigens and receptors involved in hemostasis, inflammation, angiogenesis, and immune regulation . She provided in vitro and in vivo evidence that EMP behave as vectors of bioactive molecules such as TF, EPCR, adhesive molecules, MMPs, proteases, NADPH Oxidase, thereby playing a key role in the tuning of vascular homeostasis. She also reported more recent data challenging the presumed deleterious role of EMP and providing evidence that EMP also promote cell survival, exert anti-inflammatory effects, counteract coagulation processes or induce endothelial generation. She reviewed the current mechanisms involved in EMP formation, and concluded by opening a debate on the protective or deleterious role of EMP that bring new insights into the understanding of endothelial-associated diseases. She suggested that these observations could open novel pharmacological approaches to manipulating EMP generation.
Janusz Rak (Canada) began by commenting that the terminology ‘microparticles (MP)’ and ‘microvesicles’ (MV) is often used as interchangeable descriptions of all cellular vesicles, especially in the cancer field. He provided a characterization of the major proteins, lipids and nucleic acids (DNA, mRNA, microRNA) that may be transfered horizontally between cells. Particular attention was paid to oncogenic pathways that stimulate the production of MV/MP harboring TF and that are possibly involved in thrombotic complications in patients with gliomas. Moreover, he provided data showing that the cargo of MV include several other receptors, antigens and bioactive molecules such as growth factors that are capable of stimulating tumor progression, invasion, angiogenesis and metastasis. MP from tumor cells harbor molecular information linked to cancer-related processes, and may serve as a reservoir of prognostic and predicitive biomarkers to monitor tumor progression and responses to targeted therapies
The Business Session was focused into 3 parts covering recent advances in standardization, ( part A ) new horizons raised by functional assays ( part B ), and technical challenges raised by emerging technologies ( part C ).
Part A, chaired by M.Berndt and F Dignat –George: Standardization strategies:
Nigel Key (US) presented the result on the SSC VB Working group devoted to the standardization of the pre-analytical step. The objective of the study was to compare the inter-laboratory variability using a common pre-analytical protocol (A) versus a non-standardized protocol (B). 14 laboratories participated to the workshop. Preliminary results showed that (1) a standardized protocol results in a reduction of the inter-laboratory variability in MP analysis (more markedly for flow cytometry analysis); (2) Tube volume and plasma volume that remains above the buffy layer and centrifugation conditions are major pre-analytic parameters that impact MP analysis. However, the inter-laboratory variability remains significant in some cases even when protocol A was apparently correctly applied suggesting the impact of other unidentified parameters. In this first report on the workshop, only platelet MPs (most sensitive to pre-analytic variables) or related functional properties were analyzed. The impact of pre-analytics protocols on the other MP sub-population was planned to be analyzed.
Romaric Lacroix (F) presented the progress reached on microparticle enumeration using the latest generation of flow cytometers. He showed that the new generation of flow cytometers (Gallios) displays improved FS resolution and decreased background noise compared to current device (FC500). This allows resolving previously undetectable small size MP. A new standardization protocol was proposed using calibrated beads to reproducibly measure both large and small MP. He showed that the fact to include the MP subset of small size gives access to a new information: significant correlations between old and new instruments were observed for platelet- and erythrocyte-derived MP but not for leukocyte- and endothelial-derived MP and Leu-MP. Moreover , he reported that significant difference in leukocytes-MP between patients with stable and unstable atherosclerotic plaques were only evidenced using the new standardized protocol.
PART B : Functional assays Chaired by F Dignat -George and M Berndt
Alisa Wolberg (US) described the cellular and plasma activities that regulate thrombin generation and fibrin formation, structure, and stability. A central hypothesis of her group is that MP exhibit procoagulant functions similar to their parent cells. Platelet MP (P-MP) were prepared from platelets stimulated with calcium ionophore, TRAP, or TRAP+convulxin, and monocyte and human monocytic THP-1 MP (M-MP) were prepared from cells stimulated with lipopolysaccharide. Isolated MP were assessed by transmission electron microscopy, nanoparticle tracking analysis, flow cytometry, tissue factor (TF) activity, prothrombinase activity, thrombin generation, and clot formation, density, and stability. M-MP and P-MP exhibited similar shapes and diameters. M-MP expressed functional TF, supported prothrombinase activity, and triggered shorter thrombin generation lag times than buffer controls. Compared to controls, M-MP supported faster fibrin formation, higher fibrin network density, and higher clot stability in the presence of tissue plasminogen activator. In contrast, P-MP did not exhibit TF activity and supported lower prothrombinase activity than M-MP. P-MP supported contact-dependent thrombin generation, but did not independently increase fibrin network density or stability. Interestingly, P-MP increased rates of thrombin generation and fibrin formation when mixed with THP-1-derived MPs. It was suggested that these data indicate that M-MP and P-MP differentially modulate thrombin generation and fibrin formation, structure and stability, suggesting unique contributions to thrombosis.
Philippe Poncelet (F) proposed a novel assay for measuring the plasmin generation of MP. The objective was to replace the classical method based on high speed centrifugation to purify MP from plasma. This method was time consuming and no reproducible. The new prototype assay extracts MP from plasma by immune-magnetic separation to measure the MP-linked plasmin generation in plasma samples. Higher recovery and better reproducibility were found for the new method compared to centrifugation-based method. Using this test, he showed that plasma from healthy donors display a very low activity while samples from intensive care unit patients have highly variable and sometimes dramatically elevated levels of plasmin generation. He concluded that the new assay presents better characteristics to study the relevance of this MP-dependent activity in human pathologies.
PART C : Novel methodologies, chaired by Nigel Key and Michael Berndt
Don Gabriel (US) provided an update on ISADE (Invitrox Surface Antigen Detection and Enumeration). ISADE is a Mie light scattering device that detects and counts MP down to a size of 0.15 microns. The resolution between MP of similar size is at least 0.01 microns. Accuracy and reproducibility appear to be excellent. At present, this technology can enumerate and size MP, but has not been adapted to allow the cellular source of MP to be distinguished. Pre-analytical variables found to affect results include the magnitude of the centrifugal field and the time of exposure to the field. High fields promote MP aggregation. Aggregated MP can then distort the size distribution and give misleading information. .Dr Gabriel presented data to illustrate that the efficiency of PMP production can vary with the choice and concentration of agonist, as well as mechanical shear. Other potentially important variables include temperature and buffer conditions. In addition, preliminary results from the MP size distribution in patients with acute liver failure were presented. Although statistical analysis has not been completed for this pilot trial of 56 patients, the number of MP seems higher in patients with acute liver failure compared to normal controls. Notably also, the presence of MP in the 0.2 to 0.26 micron size range was more prevalent in hypothermic patients with circulatory shock. Three of the 56 patients had MP expressing hepatocellular biomarkers by flow cytometry.
Edwin van der Pol (NL) presented the ability of two new, advanced methods (nanoparticle tracking analysis (Nanosight NS500) and resistive pulse sensing with a pore diameter of 1 µm (iZon qNano)), to measure the size and concentration of a standard population of human urine vesicles by comparison with two established techniques (transmission electron microscopy and flow cytometry). He showed that Nanoparticle tracking analysis and resistive pulse sensing detected a concentration of 1.4?1010 ml-1 and 3?1010 ml-1, respectively, which is 1000-fold higher than the observed concentration with flow cytometry. By combining the collected information with a model based on the Mie theory for the calibration of flow cytometers, he proposed a corrective factor of the sizing value of polystyrene beads for the standardization of MP detection with flow cytometry. He also brought some evidence that the detection of large vesicles by flow cytometry would be due to the simultaneous presence of multiple vesicles in the laser beam.
Chris Gardiner (UK) presented an update on nanoparticle tracking analysis, a technology based on the measurement of Brownian motions using the last version of a laser instrument (Nanosight). This technology measures particles with a size between 50 nm and 1 μm. Analysis of preparations of particles from human plasma indicated that there are 1-5 x 1010/mL cell-derived vesicles in healthy people, a concentration of particles that is approximately 1000-fold greater than estimates by conventional flow cytometry. Most “microparticles” are lipoproteins that are undetectable by flow cytometry approaches. However, the fact that particles other than real MPs or exosomes such as lipidic vesicles may interfere, advocates for specific labeling. Quantum dot labelling can be problematical. Bright high-affinity, specific fluorescent labels are necessary for phenotyping. Multiparameter measurement of micro/nanovesicles is achievable. NTA may be useful for determining the resolution of flow cytometry
Suzanne Osanto described the use of a combination of microfluidics and AFM to detect MPs directly in plasma. Tenfold-diluted EDTA PPP was flown through a microfluidic channel with a controlled pressure driven laminar flow to allow direct contact with anti-human CD41 antibody-coated mica. MPs bearing CD41 antigen were captured on this surface and subsequently detected by AFM operated in fluid tapping mode. The majority of the captured MPs have diameters (dsph) similar to prior results obtained with MPs immediately isolated from fresh PPP. High-speed centrifugation needed to isolate MPs did not influence the size distribution of MPs. Use of the microfluidic system also increased the efficiency of capturing MPs. This microfluidic system was applied to count PMPs in plasma from healthy donors, and was compared with a drop method. Considerably more CD41-positive MPs per 100 µm2 surface were detected when plasma was run through the microfludic system than when measured by the drop method. A linear dose-response curve between the number of CD41-positive MPs per 100 µm2 surface and the MP concentration was observed.
Given that the release of procoagulant platelet microparticles (PMP) is considered a hallmark of HIT, Christian Chatelain (BE) presented a novel assay based on PMP generation (PMPGA) for the early diagnosis of type II heparin-induced thrombocytopenia (HIT). This diagnosis of HIT remains challenging in clinical practice.
The performance of this PMPGA was compared with ELISA, Light Transmission Aggregometry (LTA), 14C-Serotonin Release Assay (SRA) and clinical outcomes. Sera or citrated-platelet-poor plasma of HIT-suspected patients (n=72) were incubated with citrated whole blood from healthy donors with/without unfractionated heparin (UH: 1 or 500 IU/ml). PMPs were quantified and characterized using a FACS Aria® flow cytometer. ELISA (PF4 Enhanced®), LTA, PMPGA, SRA and clinical outcomes data were compared by Chi-Square tests and ROC Curves.
In HIT patients, PMPs expressing phosphatidylserine (PS+) are generated following immune complex formation with low UH concentration whereas PMP rate decreases significantly in the presence of high UH concentration. It was proposed that the ratio of PMPs PS+ between low and high heparin concentrations be evaluated, with an optimal cut-off ratio of 2.0. The correlation between PMPGA and SRA is markedly more significant (p<0.0005, n=57 including 10 positive SRA) than LTA (p=0.0267, n=39) and ELISA (p=0.0022, n=58). Sensitivity and specificity of PMPGA were 70.0% and 97.7%, respectively, calculated with SRA as reference. Combining clinical outcome to biological testing, PMPGA sensitivity and specificity reached 100 and 88.9%, respectively.
Those cases that were discordant between SRA and PMPGA were analyzed by clinical outcome. Given its good correlation with 14C-SRA performance and clinical outcome, PMPGA has to be tested in larger scale studies as a potential new biological reference confirmation assay for HIT.
Closing remarks
Finally, Francoise Dignat George presented the update and perspectives of the ISTH Vascular Biology Working Group on the Measurement of microparticles by flow cytometry. Important questions were raised during previous SSC VB, namely: 1/ the possibility of standardizing MP enumeration by flow cytometry; 2/ the need for new generation flow cytometers or alternative technologies that would allow enumeration and characterization of particles of smaller sizes; 3/ study the impact of pre-analytic parameters on MP determination . Some of these questions have now been addressed.
During the ISTH SSC that took place in Vienna in 2008, a first collaborative workshop on the standardization of microparticle counts was set up to define the inter-laboratory reproducibility of PMP counts using flow cytometry. Presented in Boston in 2009, the results of this workshop have been published in the Journal of Thrombosis and Haemostasis . : J Thromb Haemost. 2010 Nov;8(11):2571-4 “Standardization of platelet-derived microparticle enumeration by FCM using calibrated beads : results of ISTH SSC. Colll workshop”: R., S. Robert, P. Poncelet, S. Glover, N.S. Key, F. Dignat-George, and all SSC participant. We thank all the investigators who helped to set up this workshop as well as those who actively participated .
In 2010, the pre-analytical phase was identified as a critical target for future standardization studies, and the Cairo SSC in VB was the opportunity to propose a new collaborative workshop on the impact of pre-analytic variables on MP determination. The first conclusions of this ongoing study have been presented in Kyoto. The results demonstrated that a standardized protocol results in a significant reduction of the inter-laboratory variability in PMP analysis that was more marked for flow cytometry. In addition, important advances were made during the Kyoto meeting indicating that: 1/ recent technological improvements maintain flow cytometry as a competitive analytical method to measure MP of smaller size; 2/ alternative technologies (NTA, DLS , ISADE, AFM, … ) open new options to enumerate MP of small size. The most critical remaining question is: How representative is this newly accessible part of the MP ‘iceberg’ for the clinically relevant biomarkers we are seeking ? Challenging these recent technical evolutions in pathological situations is a mandatory step to validate their real impact in clinical practice. This question suggests a direction for future collaborative working parties in the VB SSC that were proposed during Kyoto. The project proposal is to dedicate a new Collaborative Workshop on the capacity of new and existing methods to resolve the “clinically relevant MP” population. Using the recently defined standardized preanalytical protocol, the Core laboratory will prepare frozen aliquots of PFP from normal and pathological samples ( cancer, sepsis and CVD…), that will be aliquoted and sent to participating labs. A laboratory expert in one technology among the current (flow cytometry, procoagulant assays) and emerging options (NTA, DLS, AFM, ISADE……) will measure MP levels under blinded conditions. Thereafter, the core laboratory will collect data from each expert lab, and evaluate the respective value of each methodology in the evaluation of both normal and pathological samples.
