Detection Of Microparticles Research Articles

Human bone ashes found in the Dama de Elche (V–IV century B.C.) reveal its use as an ancient cinerary urn

Abstract The Dama de Elche figure is a polychromed stone life-size bust and is recognized as an emblematic piece of Iberian Art (V–IV century B.C.). The Dama de Elche possesses a small cavity in the back whose function has been object of several unconfirmed hypothesis since its discovery in 1897, due to the fact that no apparent indications of its former use could be found. This research has been centered on the analysis of the cavity and the search of data as to be able to confirm its former use. A superficial base-gypsum layer that covered the sculpture had been also detected on the surface of the cavity. Afterwards a recrystallization process took place due to temperature and relative humidity changes. By microscope techniques, several carbonaceous particles have been identified that were immersed inside the recrystallized superficial CaSO 4 .2H 2 O (gypsum) layer, not visible by visual inspection. The use of the bust as a cinerary urn has been verified with the detection of carbonaceous microparticles in which phosphorus and calcium rich fragments have been identified. The obtained Ca:P ratio indicates their bone origin. The comparison of these analyzed data with other fired remains from human bone samples found in a nearby Iberian archaeological site of the same date holds a very high similarity, which confirms its analogous origin. There are also slag and sinterized particles inside the cavitiy produced at high temperatures. These results are coherent with the cremation funerary rites of Iberian culture that included the setting, even partially, of the ashes inside an urn. The detection of strontium ions, side by side the carbonaceous particles inside the cavity, are related with the thermal process induced at the time of depositing the bone ashes, still hot, from the cremation ritual. In relation to the bust authenticity, these new found data support that the Dama de Elche is a piece of Iberian culture. No data was found to support that it might have been a XIX century forgery.

Detection and isolation of cell-derived microparticles are compromised by protein complexes resulting from shared biophysical parameters

AbstractNumerous diseases, recently reported to associate with elevated microvesicle/microparticle (MP) counts, have also long been known to be characterized by accelerated immune complex (IC) formation. The goal of this study was to investigate the potential overlap between parameters of protein complexes (eg, ICs or avidin-biotin complexes) and MPs, which might perturb detection and/or isolation of MPs. In this work, after comprehensive characterization of MPs by electron microscopy, atomic force microscopy, dynamic light-scattering analysis, and flow cytometry, for the first time, we drive attention to the fact that protein complexes, especially insoluble ICs, overlap in biophysical properties (size, light scattering, and sedimentation) with MPs. This, in turn, affects MP quantification by flow cytometry and purification by differential centrifugation, especially in diseases in which IC formation is common, including not only autoimmune diseases, but also hematologic disorders, infections, and cancer. These data may necessitate reevaluation of certain published data on patient-derived MPs and contribute to correct the clinical laboratory assessment of the presence and biologic functions of MPs in health and disease.

Measurement of circulating cell-derived microparticles by flow cytometry: Sources of variability within the assay

Introduction Circulating cell-derived microparticles (MPs) have been implicated in several disease processes and elevated levels are found in many pathological conditions. The detection and accurate measurement of MPs, although attracting widespread interest, is hampered by a lack of standardisation. The aim of this study was to establish a reliable flow cytometric assay to measure distinct subtypes of MPs in disease and to identify any significant causes of variability in MP quantification. Materials and Methods Circulating MPs within plasma were identified by their phenotype (platelet, endothelial, leukocyte and annexin-V positivity (AnnV+). The influence of key variables (i.e. time between venepuncture and centrifugation, washing steps, the number of centrifugation steps, freezing/long-term storage and temperature of thawing) on MP measurement were investigated. Results Increasing time between venepuncture and centrifugation leads to increased MP levels. Washing samples results in decreased AnnV + MPs (P = 0.002) and platelet-derived MPs (PMPs) (P = 0.002). Double centrifugation of MPs prior to freezing decreases numbers of AnnV + MPs (P = 0.0004) and PMPs (P = 0.0004). A single freeze thaw cycle of samples led to an increase in AnnV + MPs (P = 0.0020) and PMPs (P = 0.0039). Long-term storage of MP samples at -80° resulted in decreased MP levels. Conclusions This study found that minor protocol changes significantly affected MP levels. This is one of the first studies attempting to standardise a method for obtaining and measuring circulating MPs. Standardisation will be essential for successful development of MP technologies, allowing direct comparison of results between studies and leading to a greater understanding of MPs in disease.

An HC-PCF Fluorescence Spectrocopy for Detection of Microsphere Samples Based on Refractive Index Scaling Law

This paper illustrates an efficient fluorescence detection of micro particles using hollow-core photonic crystal fibers (HC-PCFs) by applying the refractive index (RI) scaling law. The variations in the central wavelength for different filling material indices are illustrated for most commonly available HC-PCFs that have cladding made of pure fused silica with array of air holes running along the entire length of the fiber. The proposed concept is verified by immobilizing fluorescent microsphere samples inside two HC-PCFs of different central wavelengths and the quantification of fluorescence inside the fibers is performed through spectroscopic analysis. The sensitivity has been compared for similar fiber with different dispersed media and different fibers with same dispersed medium.

Pre-analytical and analytical issues in the analysis of blood microparticles

Results of plasma microparticles (MPs) measurements reported in the literature vary widely. This is clearly not only related to the lack of well-standardised MP assays, but also to variations in pre-analytical conditions. In this review we will discuss the pre-analytical variables related to plasma and MP preparation which may affect MP analysis. Additionally we will address several analytical issues in commonly used MP assays and briefly discuss some novel approaches for the detection and characterisation of MPs. Ideally MP measurements should be performed in plasma, freshly prepared directly after blood withdrawal. As platelet contamination seems to be one of the major pre-analytical problems in processing plasma for MP measurement, the use of platelet-free plasma may be preferred. When frozen-thawed plasma is used, especially PMP and annexinV-positive MP counts should be interpreted with caution. When flow cytometry is chosen as a method for quantification of MPs, some analytical conditions should be standardised, e.g. settings of the flow cytometer, quality of the antibodies, and use of counting beads. Fluorescence-nanoparticle tracking analysis and atomic force microscopy can accurately count nanosized MPs, but unfortunately the operational procedures of both methods are still time consuming and they give no information on the functional properties of MPs. The MP-TF activity assay provides information on MPs carrying active TF, regardless of their parental origin. Ultimately, standardisation of pre-analytical procedures and the introduction of reliable and rapid methods for the measurement of MPs are urgently needed to facilitate their use as biomarker in the pathophysiology of diseases.

Optical and non-optical methods for detection and characterization of microparticles and exosomes.

Microparticles and exosomes are cell-derived microvesicles present in body fluids that play a role in coagulation, inflammation, cellular homeostasis and survival, intercellular communication, and transport. Despite increasing scientific and clinical interest, no standard procedures are available for the isolation, detection and characterization of microparticles and exosomes, because their size is below the reach of conventional detection methods. Our objective is to give an overview of currently available and potentially applicable methods for optical and non-optical determination of the size, concentration, morphology, biochemical composition and cellular origin of microparticles and exosomes. The working principle of all methods is briefly discussed, as well as their applications and limitations based on the underlying physical parameters of the technique. For most methods, the expected size distribution for a given microvesicle population is determined. The explanations of the physical background and the outcomes of our calculations provide insights into the capabilities of each method and make a comparison possible between the discussed methods. In conclusion, several (combinations of) methods can detect clinically relevant properties of microparticles and exosomes. These methods should be further explored and validated by comparing measurement results so that accurate, reliable and fast solutions come within reach.

Calcium-Independent Detection and Isolation of Phosphatidylserine-Exposing Cells and Microparticles Using Recombinant Del-1

AbstractAbstract 4224Del-1 is an extracellular matrix protein that is highly expressed during the embryonic period. It comprises, from N- to C-terminus, three epidermal growth factor (EGF) domains (containing an RGD motif in the second EGF domain) and two factor VIII-homologous C domains (C1C2 domain). After the embryonic period, hypoxia and vascular injury can increase Del-1 expression by endothelial cells and macrophages, promoting angiogenesis and phagocytosis of phosphatidylserine (PS)-exposing apoptotic bodies. This is possibly due to the capacity of Del-1 to bridge the interaction between apoptotic bodies and phagocytes. Del-1 binds to PS-exposing cells and membrane fragments through its C1C2 domains and to integrins of phagocytic cells through its RGD sequence, enhancing phagocytosis. We expressed in mammalian cells a Del-1 fragment composed of the C1 and C2 domains containing two human protein C tags and a biotin tag at the C-terminus. The recombinant fragment was purifed using monomeric avidin resin. The recombinant Del-1 C1C2 bound PS specifically on a commercial lipid array (PIP-strip, Echelon), a finding confirmed in a 96-well assay using immobilized lipids. The binding of C1C2 Del-1 to phosphatidylserine (PS) was consistent and stable when compared to the binding of commercially available annexin-V (biotinylated and PE-conjugated). We evaluated by flow cytometry the ability of Del-1 C1C2 to bind membrane-exposed PS. Apoptotic THP-1 cells were incubated with Del-1 C1C2 in the absence of calcium or with annexin V (2.5 mM calcium was present in all the buffers and through all washing steps). Protein binding was detected using a monoclonal anti-biotin-PE conjugated antibody. Neither the percentage of PS-positive cells nor the mean fluorescence intensity (MFI) was statistically different between Del-1 C1C2 and annexin V when samples were diluted with buffer (instead of washed) before flow cytometric analysis. However, both the percentage of PS-positive cells and MFI decreased by approximately 50% in the annexin V samples when the cells were washed after incubation. Cell-derived microparticles from outdated RBC units were also used to compare the utility of C1C2 Del-1 vs commercial PE-conjugated annexin V for the detection of microparticles by flow cytometry. We found a 1.4-fold average increase in the quantity of microparticles detected with C1C2 Del-1 when compared to annexin V. In addition, cell-derived microparticles can be isolated and concentrated from the Del-1 C1C2–treated sample using magnetic beads coupled to anti-protein C antibody. In conclusion, the C1C2 domain of Del-1 represents a powerful and sensitive tool for the detection and isolation of PS-exposing cells and microparticles. The calcium-independence of this reagent for PS simplifies the detection of apoptotic cells and microparticles in plasma. Moreover, the addition of multiple tags at the C-terminus allows the capture, labeling and enrichment of PS-specific microparticles by bridging C1C2-coated microparticles to magnetic beads. C1C2 Del-1 thus represents a powerful new tool for analysis in the fields of microparticle biology, thrombosis, and immunology. Disclosures:No relevant conflicts of interest to declare.

Erythrocytapheresis in Sickle Cell Disease Lead to An Elective Reduction of Erythrocytes-Derived Blood Microparticles

AbstractAbstract 2657 Introduction:Erythocytapheresis is a highly effective procedure used in the management of both acute and chronic complications of sickle cell disease (SCD). Indeed, exchange transfusion is recommended as a part of the initial treatment of vaso-occlusive stroke in children. Red cells exchange can also be proposed in primary and secondary stroke prevention in order to maintain a low level of HbS. Microparticles (MPs) are small vesicles released from cells after activation or apoptosis. Microparticles number is increased in SCD both at the steady state and during crisis. The majority of these microparticles are originated from erythrocytes and platelets. In SCD, erythrocytes MPs are strongly correlated with hemolysis and activation of coagulation.In this study, we aimed to measure the level of total, platelets-derived and erythrocytes-derived MPs in patients with SCD undergoing erythrocytapheresis; we compared the level of MPs before and after red cells exchange in order to evaluate if the reduction of stroke observed in patients undergoing erythrocytapheresis could be due to a specific decrease in erythrocytes-derived MPs known to be implicated in coagulation activation in this disease. Materials and Methods:After informed consent, 24 samples were harvested on a tube containing buffered sodium citrate from 22 patients with SCD undergoing erythrocytapheresis. For each patient, a sample was collected before and just after the exchange. 20 samples from donor with neither anemia nor coagulation disorders were used as controls. Each sample was centrifugated at 1500g during 15 mn, the supernatant was collected and centrifugated 2mn at 13000g at 20°C and then frozen at -80°C until analysis. For MPs detection, 38mL of the thawed supernatant was labelled with 5mL of 10X Annexine V buffer, 1mL of Annexine V-FITC, 1 mL of anti-GPA-PE and 5 mL of anti-CD41-APC. 5mL of calibrated beads at known concentration were added to 345 mL of 1X Annexine buffer just before analysis on the MoFlow cytometer (Beckman Coulter). The Megamix beads system was used in order to delimitate the FSC/SSC gate of MPs. Results:Total number of plasma MPs (1755/mL vs 273), erythrocytes-derived MPs (174/mL vs 8) and platelets-derived MPs (1520/mL vs 236) were significantly increased in SCD patients in comparison with controls. For SCD patients undergoing erythrocytapheresis, the mean HbS level was respectively 52% and 24% before and after red cells exchange. No significant difference in the total hemoglobin concentration was noted. The mean number of MPs did not differ before and after the exchange (1755/mL versus 1940/mL respectively, student test p>0.05). The mean number of platelets-derived MPs remained stable before and after the exchange (1520/mL versus 1250mL, student test, p>0.05). In sharp contrast, we observed a significant clearance of the erythrocytes-derived MPs in the plasma of SCD patients after red cells exchange (mean number of GPA+-MPs: 174/mL before exchange versus 77mL after, student test, p<0.05). However, we could also observe a great variation of the number of erythrocytes and platelets-derived MPs between the different patients. Conclusion:The number of erythrocytes-derived MPs decreased specifically after red cells exchange, in contrast with the total and the platelets-derived MPs which remained stable; this selective decrease in the GPA+-MPs of SCD patients could be due to a reduction in the total number of red cells containing a high HbS content in the circulation, because of their replacement by normal erythrocytes that have a far lower capacity to produce MPs. It has been recently shown that in SCD, GPA+-MPs are specifically correlated with coagulation and fibrinolysis activation, underlying the fact that a direct link exists between hemolysis, erythrocytes- derived MPs liberation and procoagulant state. The specific clearance of the GPA+-MPs after red cells exchange could partially explain the positive effects of this procedure in primary and secondary prevention of stroke in SCD. However, that GPA+-MPs remained statistically increased after red cells exchange in comparison with controls; whether a low number of post-exchange MPs could be correlated with a better outcome is actually under investigation. Disclosures:No relevant conflicts of interest to declare.

Overcoming Limitations of Microparticle Measurement by Flow Cytometry

Circulating microparticles are submicron vesicles released from cell membranes in response to activation or apoptosis. Acknowledgment of their role both as markers and pathogenic effectors in thrombosis, inflammation, and the spread of cancer has increased the interest of their measurement in clinical practice. However, assessment of their clinical use is impeded by technological issues. Among the different methodologies available, flow cytometry is the most commonly used technique. This review addresses flow cytometry limitations in microparticle measurement that may be subdivided into three domains: sizing, probing, and counting. This article also covers the various standardization strategies and technological improvements that have been proposed to overcome these limitations. New tools using size-calibrated beads and recent progress in instrumentation have opened new avenues to improve detection of microparticle populations of smaller sizes. Significant optimization in microparticle detection is also expected from the use of new fluorescent dyes and the establishment of practical recommendations. Finally, absolute counting of microparticles will also benefit from adapted bead-based strategies or, alternatively, from the generalized availability of volumetric systems. Overall, recent technological improvements maintain flow cytometry as a highly competitive analytical method to measure microparticles. Challenging these evolutions in pathological situations is a mandatory step to validate their real impact in clinical practice.

Microparticles in physiological and in pathological conditions

Chronic diseases pose a severe burden to modern National Health Systems. Individuals nowadays have a far more extended lifespan than in the past, but healthy living was only scantily extended. As much as longer life is desirable, it is saddened by chronic diseases and organ malfunctions. One contributor to these problems was recognized to be represented by microparticles (MPs). Our purpose is to better understand MPs, to contrast their ominous threat and possible clinical importance. For this intent we correlated MPs with thrombotic pathologies, hemophilia, malaria, diabetes, cardiovascular diseases, endothelial dysfunctions, pulmonary hypertension, ischemic stroke, pre-eclampsia, rheumatologic diseases-rheumatoid arthritis, polymyositis-dermatomyositis, angiogenesis and tumor progression-cancer; we listed the possibilities of using them to improve transfusion methods, as a marker for acute allograft rejection, in stem cell transplantation, as neuronal biomarkers, to understand gender-specific susceptibility for diseases and to improve vaccination methods and we presented some methods for the detection of MPs.

Differential diagnosis of acute rejection and chronic cyclosporine nephropathy after rat renal transplantation by detection of endothelial microparticles (EMP)

Endothelial microparticles (EMP) are small vesicles smaller than 1.0μm, released from endothelial cells (EC) during their activation and (or) apoptosis. The assay of the level of elevated EMP is a new approach to evaluate the dysfunction of endothelial cell. EMP can be classified into several types according to their membrane molecular, and the levels of various types of EMP may be different. As the most cost-effective immunodepressant, cyclosporine A (CsA) has been used widely in organ transplantation. But its dose is hard to control, under-medication may cause the acute rejection (AR) and overdose may cause chronic cyclosporine nephropathy (CCN). The cyclosporine A (CsA) caused CCN and the AR caused renal injury after renal transplantation are both vascular diseases related with endothelial dysfunction, and up to now, there is still no effective method to distinguish the two kinds of diseases. Owing to distinct pathogenesis of the two kinds of vascular diseases, the level of each type of EMP originated from vascular endothelial cells may be different. We hypothesize that maybe we can distinguish them by detecting the different levels of some types of EMP which is also related with vascular disease, and we propose to prove our hypothesis through animal experiment. If our hypothesis is proved, it will be more helpful for clinicians to adjust the dose of CsA promptly according to the differential diagnosis of the two kinds of diseases.

A novel broadband impedance method for detection of cell-derived microparticles

A novel label-free method is presented to detect and quantify cell-derived microparticles (MPs) by the electrochemical potential-modulated electrochemical impedance spectroscopy (EIS). MPs are present in elevated concentrations during pathological conditions and play a major role in the establishment and pathogenesis of many diseases. Considering this, accurate detection and quantification of MPs is very important in clinical diagnostics and therapeutics. A combination of bulk solution electrokinetic sorting and interfacial impedance responses allows achieving detection limits as low as several MPs per μL. By fitting resulting EIS spectra with an equivalent electrical circuit, the bulk solution electrokinetic and interfacial impedance responses were characterized. In the bulk solution two major relaxations were prominent—β-relaxation in low MHz region due to the MP capacitive membrane bridging, and α-relaxation at ∼10 kHz due to counter ions diffusion. At low frequencies (10–0.1 Hz) at electrochemical potentials exceeding −100 mV, a facile interfacial Faradaic process of oxidation in MPs coupled with diffusion and non-Faradaic double layer charging dominate, probably due to oxidation of phospholipids and/or proteins on the MP surface and MP lysis. Buffer influence on the MP detection demonstrated that a relatively low conductivity Tyrode's buffer background solution is preferential for the MP electrokinetic separation and characterization. This study also demonstrated that standard laboratory methods such as flow cytometry underestimate MP concentrations, especially those with smaller average sizes, by as much as a factor of 2–40.

Circulating endothelial progenitor cells, microparticles and vascular disease

Circulating endothelial progenitor cells, microparticles and vascular disease

Circulating endothelial cells, microparticles, and markers of inflammation and coagulation in glioblastoma patients before and after protracted temozolomide and radiotherapy.

2086 Background: Circulating endothelial cells (CECs) have prognostic and predictive value in breast cancer patients treated with metronomic chemotherapy. High levels of microparticles (MPs) are associated to shorter survival in prostate cancer. We investigated the pre- and posttreatment levels of CECs, MPs and several inflammation and coagulation markers in patients with newly diagnosed glioblastoma (GB) who received protracted temozolomide (PTMZ) and radiotherapy (RT). Methods: Peripheral blood was obtained from 14 patients with GB before and 4-5 weeks after beginning of treatment. Healthy subjects (n = 22) were used as controls. Isolation and quantification of CECs were determined by an immunomagnetic technique using CD146 and lectin-1 of Ulex Europaeus combined with fluorescein. Flow cytometry of plasma was used for detection of total phosphatidylserine-positive MPs, labeled with FITC-Annexin V. Endogen thrombin generation (ETG) was measured by the calibrated automated thrombogram method (CAT). Prothrombin fragment 1+2 (F1+2) and interleukin-6 (IL-6) were measured by ELISA. Results: Mean number of CECs was significantly increased in patients before (112 ± 65 cells/mL) and after treatment (122 ± 80 cell/mL) as compared to controls (10 ± 5 cell/mL; p < 0.0001). Mean number of MPs was significantly increased in patients before (794 ± 573 MPs/μ L) and after treatment (408 ± 207 MPs/μ L) as compared to controls (234 ± 174 MPs/μ L; p < 0.05). Levels of IL-6 were significantly higher in patients before and after treatment (IL6, 8.3 ± 14.3 pg/mL; 3.2 ± 3.1 pg/mL) than in controls (0.9 ± 0.8 pg/mL). Levels of coagulation markers were significantly higher in patients before treatment (F1+2, 0.33 ± 0.18 pmol/mL; ETG, 234 ± 106 nM). However, levels of both markers decreased near normal values (F1+2, 0.22 ± 0.07 vs. 0.17 ± 0.04 pmol/mL; ETG, 169 ± 146 vs. 162 ± 72 nM) after treatment. Conclusions: CECs and MPs, as well as a set of markers of inflammation and coagulation, are strongly elevated in patients with GB. Coagulation markers decrease after treatment with PTMZ+RT. The influence of treatment on CEC and MP levels is unclear and needs further study. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Schering-Plough Schering-Plough

Medicaments: Gifts from the jungle

Medicaments: Gifts from the jungle

Flow cytometric detection of endothelial microparticles (EMP): Effects of centrifugation and storage alter with the phenotype studied

Introduction Endothelial microparticles (EMP) are released into the circulation in case of endothelial disturbance, and are therefore increasingly investigated as a biomarker reflecting disease activity. Numerous pre-analytic methods have been proposed for their flow cytometric enumeration, but standardization is still lacking. In this study we evaluated the influence of centrifugation and storage conditions on EMP quantification. Materials and Methods Platelet-poor plasma (PPP) from 10 healthy volunteers was prepared by centrifugation at 1 550 g for 20 minutes twice. A first aliquot of PPP was analyzed immediately, a second after storage at 4 °C for 7 hours. A third and fourth aliquot were snap-frozen and stored at -80 °C for 7 and 28 days. A final aliquot was further centrifuged at 10 000 g for 10 minutes and analyzed immediately. EMP were defined as CD31+CD42b-, CD62E+, CD144+ or CD144+CD105+ particles, smaller than 1.0 µm. Results High speed centrifugation led to a significant loss of CD31+CD42b- EMP (p = 0.004). A good correlation between PPP and high speed centrifuged PPP was only found for CD144+ EMP (Kendall tau b = 0.611, p = 0.025). Storage at 4 °C did not affect EMP quantification. However, freezing at -80 °C increased CD31+CD42b- and CD62E+ EMP counts, and lowered CD144+ EMP (p < 0.05). Nevertheless, the agreement among the different storage conditions was relatively good (Kendall coefficient of concordance > 0.487; p < 0.05). Conclusion The flow cytometric detection of EMP varies with the centrifugation protocol and the storage method used, and these changes also depend on the phenotype studied. The results of this study caution against comparing study results gathered with different EMP laboratory protocols.

Use of SYTO 13, a fluorescent dye binding nucleic acids, for the detection of microparticles in in vitro systems

Microparticles (MPs) are small membrane-bound vesicles that are released from activated or dying cells by a blebbing process. These particles contain nuclear and cytoplasmic components and represent unique biomarkers for disease. The small size of particles, however, limits detection using flow cytometry with either light scatter or staining for surface markers. Because MPs contain DNA and RNA, we have explored the use of SYTO 13, a member of the class of SYTO dyes, for particle detection. SYTO 13 is cell permeable and has a high fluorescent yield when bound to DNA or RNA. In this study, we compared detection of MPs using either light scatter or SYTO 13 staining, testing the hypothesis that, with fluorescence detection with SYTO 13, problems of "noise" with light scatter are reduced and the range of MP sizes detected is increased. In these experiments, particles were obtained from lymphoid cell lines treated in vitro to undergo apoptosis. As these results showed, STYO 13 allowed the detection of 1.5-2.9 times as many particles as did light scatter. The increased sensitivity was observed with three different cell lines and was independent of inducing stimulus. Treatment of fixed and permeabilized MPs with DNase and RNase showed that SYTO 13 binding resulted from interaction with both DNA and RNA. Together, these findings indicate that the nucleic acid content of MPs provides the basis for their detection in in vitro systems and suggests the utility of fluorescent dyes like SYTO 13 for more sensitive quantitative assays.

Detection of circulating microparticles by flow cytometry: influence of centrifugation, filtration of buffer, and freezing

The clinical importance of microparticles resulting from vesiculation of platelets and other blood cells is increasingly recognized, although no standardized method exists for their measurement. Only a few studies have examined the analytical and preanalytical steps and variables affecting microparticle detection. We focused our analysis on microparticle detection by flow cytometry. The goal of our study was to analyze the effects of different centrifugation protocols looking at different durations of high and low centrifugation speeds. We also analyzed the effect of filtration of buffer and long-term freezing on microparticle quantification, as well as the role of Annexin V in the detection of microparticles. Absolute and platelet-derived microparticles were 10- to 15-fold higher using initial lower centrifugation speeds at 1500 × g compared with protocols using centrifugation speeds at 5000 × g (P < 0.01). A clear separation between true events and background noise was only achieved using higher centrifugation speeds. Filtration of buffer with a 0.2 μm filter reduced a significant amount of background noise. Storing samples for microparticle detection at −80°C decreased microparticle levels at days 28, 42, and 56 (P < 0.05 for all comparisons with fresh samples). We believe that staining with Annexin V is necessary to distinguish true events from cell debris or precipitates. Buffers should be filtered and fresh samples should be analyzed, or storage periods will have to be standardized. Higher centrifugation speeds should be used to minimize contamination by smaller size platelets.

A multicolor flow cytometric assay for measurement of platelet-derived microparticles

Introduction Flow cytometry (FCM) is the most commonly used method for detection of platelet-derived microparticles (PDMPs), but it is poorly standardized and mainly used for “bedside” analyses in fresh samples. If PDMPs could be analyzed in previously frozen samples it would increase the usefulness of the method. However, cell membrane fragments from contaminating cells created during freezing/thawing may cause artifacts and disturb measurements. Materials and Methods PDMPs were labeled with monoclonal antibodies directed against CD42a and CD62P, or CD42a and CD142. The PDMP gate was determined using forward scatter (FSC) and CD42a expression. The mean fluorescence intensities (MFIs) of CD62P or CD142 positive particles were translated into MESF -values (Molecules of Equivalent Soluble Fluorochrome) using a standard curve. FITC-labeled phalloidin (which binds to intracellular actin) was used to detect destroyed cells/cell fragments. Results Phalloidin-positive particles were significantly more common in supernatants of frozen/thawed platelet rich and platelet poor plasma samples compared with supernatants of platelet free plasma. High-speed centrifugation was then used to obtain PDMP samples with low contamination of cell fragments. Electron microscopy showed that these samples contained numerous round stained particles with cellular membranes of a size of 100–700 nm. Reproducibility experiments using plasma samples from healthy individuals showed that the coefficients of variation (CVs) of MESF values of CD62P and CD142 (both intra- and interassay) were < 10%, and the variation between two cytometers in two different laboratories was < 5%. We also found that PDMP expression of CD142 (i.e. tissue factor [TF]) and CD62P (i.e P-selectin) was around two times higher in samples from type 1-diabetes patients compared with those from healthy controls ( p < 0.001). Conclusions The use of MESF values to quantify PDMP expression of P-selectin and TF yields reproducible data and enables comparison of data between laboratories. If high-speed centrifugation is performed, contamination of cell fragments is low in frozen/thawed samples.

Cell‐derived microparticles: a new challenge in neuroscience

Microparticles (MPs) are membrane fragments shed by cells activated by a variety of stimuli including serine proteases, inflammatory cytokines, growth factors, and stress inducers. MPs originating from platelets, leukocytes, endothelial cells, and erythrocytes are found in circulating blood at relative concentrations determined by the pathophysiological context. The procoagulant activity of MPs is their most characterized property as a determinant of thrombosis in various vascular and systemic diseases including myocardial infarction and diabetes. An increase in circulating MPs has also been associated with ischemic cerebrovascular accidents, transient ischemic attacks, multiple sclerosis, and cerebral malaria. Recent data indicate that besides their procoagulant components and identity antigens, MPs bear a number of bioactive effectors that can be disseminated, exchanged, and transferred via MPs cell interactions. Furthermore, as activated parenchymal cells may also shed MPs carrying identity antigens and biomolecules, MPs are now emerging as new messengers/biomarkers from a specific tissue undergoing activation or damage. Thus, detection of MPs of neurovascular origin in biological fluids such as CSF or tears, and even in circulating blood in case of blood-brain barrier leakage, would not only improve our comprehension of neurovascular pathophysiology, but may also constitute a powerful tool as a biomarker in disease prediction, diagnosis, prognosis, and follow-up.