Evaluation of microparticles in whole blood by multicolour flow cytometry assay

Christersson, Christina; Johnell, Matilda; Siegbahn, Agneta

doi:10.3109/00365513.2013.769278

Cited by 38 publications

(30 citation statements)

References 58 publications

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“…Our study not only indirectly supports the accumulating evidence that MPs are markers of an increased risk of cardiovascular events in high-risk patients [40,41,42,43] but also partly clarifies the mechanisms of how the increased MP levels from ACS patients further damage the vascular function in vitro. These findings may further confirm the question of whether MPs can be used as biomarkers for the early detection of cardiovascular events, as already suggested by others [8].…”

Section: Discussionsupporting

confidence: 70%

Microparticles from Patients with the Acute Coronary Syndrome Impair Vasodilatation by Inhibiting the Akt/eNOS-Hsp90 Signaling Pathway

Han

Chang²,

Wang

et al. 2015

Cardiology

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Objectives: Endothelial dysfunction is involved in the development of the acute coronary syndrome (ACS). Plasma microparticles (MPs) from other diseases have been demonstrated to initiate coagulation and endothelial dysfunction. However, whether MPs from ACS patients impair vasodilatation and endothelial function remains unclear. Methods: Patients (n = 62) with ACS and healthy controls (n = 30) were recruited for MP isolation. Rat thoracic aortas were incubated with MPs from ACS patients or healthy controls to determine the effects of MPs on endothelial-dependent vasodilatation, the phosphorylation of Akt and endothelial nitric oxide synthase (eNOS), the interaction of eNOS with heat shock protein 90 (Hsp90), and nitric oxide (NO) and superoxide anion (O₂^-) production. The origin of MPs was assessed by flow cytometry. Results: MP concentrations were increased in patients with ACS compared with healthy controls. They were positively correlated with the degree of coronary artery stenosis. MPs from ACS patients impair endothelial-dependent vasodilatation, decrease both Akt and eNOS phosphorylation, decrease the interaction between eNOS and Hsp90, and decrease NO production but increase O₂^- generation in rat thoracic aortas. Endothelial-derived MPs and platelet-derived MPs made up nearly 75% of MPs. Conclusions: Our data indicate that MPs from ACS patients negatively affect endothelial-dependent vasodilatation via Akt/eNOS-Hsp90 pathways.

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Section: Discussionsupporting

confidence: 70%

Microparticles from Patients with the Acute Coronary Syndrome Impair Vasodilatation by Inhibiting the Akt/eNOS-Hsp90 Signaling Pathway

Han

Chang²,

Wang

et al. 2015

Cardiology

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“…Prior studies have reported that delays in processing the sample can lead to increases in platelet and annexin V1 microvesicles (Table 3) (12)(13)(14)(15)(16). A 1-h delay resulted in about a 50 2 100% increase in platelet and annexin V1 microvesicle counts with longer delays associated with larger increases in microvesicle counts.…”

Section: Sample Transportation and Processingmentioning

confidence: 99%

“…To prepare platelet free plasma the sample is typically spun twice at 2,000g for 15 min, a total of 60,000g-min of sedimentation, 60 times more than the preparation of PRP. This additional centrifugation of whole blood or platelet rich plasma removes cells and platelets but also reduces red cell, platelet, and annexin V1 microvesicles to a variable extent from sample to sample (16,17,20). Figure 1 shows total light scatter particles and platelet-derived (CD411) microvesicles in platelet rich plasma versus platelet microvesicles in platelet free plasma.…”

Section: Centrifugationmentioning

confidence: 99%

Measurement of microvesicle levels in human blood using flow cytometry

Chandler

2016

Cytometry Part B Clinical

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Microvesicles are fragments of cells released when the cells are activated, injured, or apoptotic. Analysis of microvesicle levels in blood has the potential to shed new light on the pathophysiology of many diseases. Flow cytometry is currently the only method that can simultaneously separate true lipid microvesicles from other microparticles in blood, determine the cell of origin and other microvesicle characteristics, and handle large numbers of clinical samples with a reasonable effort, but expanded use of flow cytometric measurement of microvesicle levels as a clinical and research tool requires improved, standardized assays. The goal of this review is to aid investigators in applying current best practices to microvesicle measurements. First pre-analytical factors are evaluated and data summarized for anticoagulant effects, sample transport and centrifugation. Next flow cytometer optimization is reviewed including interference from background in buffers and reagents, accurate microvesicle counting, swarm interference, and other types of coincidence errors, size calibration, and detection limits using light scattering, impedance and fluorescence. Finally current progress on method standardization is discussed and a summary of current best practices provided. V C 2016 Clinical Cytometry Society

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“…However, PDMP assays may consist of quantifying assays, functional assays and morphologic assays. Although the ISTH report was a first step towards achieving standardization of assay methods for evaluating PDMPs, studies continue to use different assay types [24][25][26][27][28][29][30][31][32] . Strasser and coworkers showed a good correlation among three different methods of characterizing PDMPs and suggested that an analysis method other than FCM should be used concurrently to detect MPs when the PDMP size is below the limit of detection on FCM 24) .…”

Section: Discussionmentioning

confidence: 99%

“…Obtaining an "accurate" evaluation of PDMPs is difficult due to many concerns. Moreover, the size, roles and concentrations of PDMPs in the plasma of healthy individuals have not been adequately determined 29,33) . Therefore, PDMP analyses should be interpreted with caution, taking into account the pitfalls of each method and purpose of the specific clinical investigation.…”

Section: Max Pdmp/plts Ratio For Predicting Hospital Mortalitymentioning

confidence: 99%

Association of the Plasma Platelet-Derived Microparticles to Platelet Count Ratio with Hospital Mortality and Disseminated Intravascular Coagulopathy in Critically Ill Patients

Ohuchi

Fujino

Kishimoto

et al. 2015

JAT

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Aim:The role of platelet-derived microparticles (PDMPs) in the crosstalk between coagulopathy and inflammation in critically ill patients remains unclear. The aim of this cohort observational study was to investigate the associations between the PDMP levels and hospital mortality or disseminated intravascular coagulopathy (DIC). Methods: This study included 119 patients who were admitted to the ICU. The PDMP levels were measured using an enzyme-linked immunosorbent assay three times a week, for a total of 372 samples. We calculated the maximum (max) PDMP value, max PDMP/platelet (PDMP/Plts) ratio (converted to the PDMP levels per 10 4 platelets) and nadir platelet count during the ICU stay. Baseline patient data and scores, including the Japanese Association for Acute Medicine (JAAM) DIC score, were collected, and potential predictors were analyzed for possible associations with hospital mortality. Results: The max PDMP/Plts ratio was significantly different comparing the survivors (n 98: median, 2.54) and non-survivors (n 21: median 17.59; p 0.001). There was a weak but statistically significant negative correlation between the max PDMP level and nadir platelet count (r 0.332, p 0.001). The max PDMP level and max PDMP/Plts ratio were higher in the DIC group (81.48 and 9.27, respectively) than in the non-DIC group (34.88 and 2.35, p 0.001 and p 0.001, respectively). The max PDMP/Plts ratio was the only variable found to be independently associated with hospital mortality according to a multivariate logistic regression analysis. Conclusions: PDMPs are involved in the development of DIC but are not related to hospital mortality. There is a good association between the PDMP/Plts ratio and hospital mortality and/or DIC in critically ill patients.

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Evaluation of microparticles in whole blood by multicolour flow cytometry assay

Abstract: This multicolour flow cytometry assay in whole blood mimics the in vivo situation by avoiding several procedure steps interfering with the MP count. By standardized quantification of MPs a reference interval of MPs can be created.

Cited by 38 publications

References 58 publications

Microparticles from Patients with the Acute Coronary Syndrome Impair Vasodilatation by Inhibiting the Akt/eNOS-Hsp90 Signaling Pathway

Microparticles from Patients with the Acute Coronary Syndrome Impair Vasodilatation by Inhibiting the Akt/eNOS-Hsp90 Signaling Pathway

Measurement of microvesicle levels in human blood using flow cytometry

Association of the Plasma Platelet-Derived Microparticles to Platelet Count Ratio with Hospital Mortality and Disseminated Intravascular Coagulopathy in Critically Ill Patients

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