Microparticles In Peripheral Blood Research Articles

Comparative Evaluation of CD34+/CD33+/CD3+/CD19+ Microparticles in Peripheral Blood of Patients during Allogeneic Stem Cell Transplantation

Introduction Allogeneic hematopoietic cell transplantation (allo-HCT) has been widely used as a potential curative strategy of hematological malignancies. In principle, the conditioning and the immunological response of the graft are basic mediators of the procedure. Cell-derived microparticles (MPs) are small membrane vesicles considered as markers of cell activation and apoptosis. Recently it was shown that they participate in important biological functions at pathological conditions associated mainly with endothelial injury, thrombosis and inflammation. The potential role of MPs from hematopoietic cells during HCT has not been addressed yet. In our previous work we have shown that CD34+ cells from cord blood transplants release MPs that carry microRNAs. The aim of this study was to quantify the MPs of CD34+/CD33+/CD19+/CD3+ cells in the peripheral blood of patients undergoing allo-HCT, compare them with the MPs of the graft that they have received and investigate their relationship with various parameters.Methods Samples were collected from the graft and from the peripheral blood of 7 patients who underwent allo-HCT in our department. Four patients were diagnosed with acute myeloid leukemia (AML) and 3 with acute lymphoblastic leukemia (ALL). Informed consent was obtained from all patients. Patients received mobilized blood stem cell transplant from sibling donors. All patients were at complete remission before allo-HCT. Regarding the conditioning 5 patients received myeloablative regimen and 2 reduced intensity conditioning (RIC). All patients achieved successful and prompt engraftment. The MPs were isolated after centrifugation of the plasma and immunophenotyped by four-color fluorescence using antibodies against CD34, CD33, CD19 and CD3 as well as Annexin V (AnnV+). The number of MPs has been estimated in the graft as well as in the peripheral blood of the patients at days 0, 4 and 14 (day0, +4d, +14d) from the transplantation. Statistical analysis was performed using t-test, Pearson's and Spearman's depending on the normality of the distribution of variables. Alterations in the number of MPs after day0 might be the result of the conditioning's effect and regarding the CD34+ MPs might be a correlation with the engraftment.Results Myeloid-derived MPs(CD33+MPs) were decreased significantly from day0 to +14d (p<0,019,Spearman's rho =0,836) as well at +4d to +14d (p<0,049,Spearman's rho =0,757). Interestingly, stem cell derived-MPs (CD34+MPs) were significantly associated with B-lymphocyte-derived MPs (CD19+MPs) at +4d and +14d respectively (p<0,007,Spearman's rho =0,890; p<0,001,Spearman's rho =0,944). CD3+cells correlated significantly with CD3+MPs at +14d(p<0,034, Spearman's rho=0,791). The number of all measured AnnV+/CD34+/CD33+/CD19+/CD3+ MPs at +d14 was decreased compared to the number of AnnV+/CD34+/CD33+/CD19+/CD3+ MPs at day0 and +4d.Conclusions In this study we have identified and monitored hematopoietic stem cell and MPs in the peripheral blood of patients undergoing allo-HCT. We have to mention that the number of MPs at day 0 represents the profile of the patient immediately after conditioning and before receiving the graft. The possible role of MPs in the engraftment could serve as a valuable parameter. DisclosuresNo relevant conflicts of interest to declare.

Lack of CD34 produces defects in platelets, microparticles, and lung inflammation.

Lung innate immune activation results in acute lung inflammation, which is characterized by alveolar barrier disruption and accumulation of cellular lung aggregates comprising neutrophils, platelets, mononuclear cells, and microparticles. CD34 is a sialomucin, with pan-selectin affinity and recently shown to protect the endothelial barrier in a bleomycin-induced lung injury model. However, there is very little information about the fundamental role of CD34 in regulation of the lung innate immune response. We hypothesized that CD34 regulates leukocyte recruitment by promoting optimal platelet activation (aggregation and spread) during bacterial lipopolysaccharide (LPS)-induced acute lung injury. Therefore, we utilized CD34 knock-out (KO) and wild-type (WT) mice to analyze and compare the morphology and expression of leukocyte subsets from the pulmonary and systemic compartments. We utilized the chemotactic N-formylated tri-peptide, fMLP, to understand platelet aggregation in vitro, and the fundamental immune stimulant, LPS, to induce lung injury and understand platelet activation ex vivo. Our data reveal that under steady-state conditions, KO mice possess large aggregates of integrin β3 (CD61)-positive microparticles in peripheral blood. Moreover, the KO mice recruit a large number of neutrophils to lungs, which are not cleared even at 36-h post-LPS exposure. The KO mice display an increased platelet CD61 expression, which aggregates, but does not spread normally in response to in vitro fMLP treatment. The KO platelets display similar deficits in their spreading ability even after ex vivo LPS exposure. Thus, our data demonstrate that CD34 modulates platelet biology, microparticle aggregation, and neutrophil recruitment during murine lung inflammation.

Relevance of elevated microparticles in peripheral blood and synovial fluid of patients with rheumatoid arthritis

Introduction: Microparticles (MPs) are submicron sized heterogeneous membrane-bound vesicles released from cells undergoing cell activation or apoptosis. Elevated platelet-derived MPs (PMPs) are reported in rheumatic diseases. We profiled Annexin-V+ MPs and CD61+ MPs in plasma and synovial fluid of patients with rheumatoid arthritis (RA) and their clinical correlates. Methods: Twenty-three newly diagnosed disease-modifying anti-rheumatic drug naive patients with RA, 17 with osteoarthritis (OA) and 22 healthy controls (HC) were enrolled. Five milliliters of synovial fluid from the knee joint of patients with RA and OA was collected; 5 ml of peripheral blood was collected from patients with RA, OA, and HC. Cell-free synovial fluid and platelet poor plasma samples were stained with Annexin-V-APC and Anti-CD61-BV510 antibodies followed by flow cytometry analysis (FACSAria III) (results expressed as the mean ± standard deviation of % population). Results: Significantly greater levels of synovial fluid MPs (Annexin-V+ MPs) were observed in RA versus OA (P Conclusions: Higher levels of Annexin-V+ MPs in both plasma and synovial fluid of RA suggest a role for MPs in the pathogenesis of RA. Elevated plasma PMPs (Annexin V+ CD61+ MPs) in RA may suggest their role in systemic involvement. Furthermore, increased levels of MPs other than those of platelet origin (Annexin V+ CD61− MPs) indicate the necessity to study the MPs from other cell lineages.

Bone Marrow Contains High Levels of Microparticles

Background: Microparticles (MP) are shed in humans from a broad variety of cells and play an important role in activation of coagulation, cell to cell interaction and transport of membrane components. Alternations in plasma levels have been found in a variety of diseases, mostly associated with thrombosis and inflammation. However, little is known about tissue levels of MP. The aim of this pilot study was to investigate the bone marrow compartment as a possible source of circulating MP.Material and Methods: MP were analysed in bone marrow blood and peripheral blood in 12 healthy stem cell donors. MPs were isolated and measured by flow-cytometric double-staining (FACScalibur, BD) with Anexin V and cell-specific antibodies for hematopoietic cells, platelets, white blood cells, red blood cells and endothelial cells. Statistical analysis was performed with SPSS for Windows 20.0.Results: Total MP levels differed between bone marrow and peripheral blood: 14.8 x109/l [8.5–19.3] vs. 9.2 x109/l [3.8–14.8] (median value [25-75 percentile]), p=0.060. Pattern of MP´s origin varied likewise: in bone marrow blood (bmb), main cell source of MP were CD235a positive red blood cells/erythropoetic cells (6.4x109/l [3.7–13.7], 43.2% of total bmb MP), CD61 positive platelets/megacaryocytes (4.1x109/l [1.7–10.1], 27.7% of total bmb MP), CD45 positive leucocytes/myeloic and lymphatic progenitor cells (3.8x109/l [2.7-4.9], 25.7% of total bmb MP) and CD62e positive endothelial cells (0.3x109/l [0.1-1.0], 2.0% of total bmb MP). In contrast, MP in peripheral blood (pb) mainly derived from CD61 positive platelets/megacaryocytes (8.5x109/l [2.4–14.4], 92.0% of total pb MP), CD45 positive leucocytes/myeloic and lymphatic progenitor cells (0.5x109/l [0.2–1.1], 4.5% of total pb MP), CD235a positive red blood cells/erythropoetic cells (0.2x109/l [0.1–0.6], 1.8% of total pb MP), and CD62e positive endothelial cells (0.1x109/l [0.0–0.1], 0.9% of total pb MP). Mean levels of tissue factor bearing MP were low in bone marrow blood as well as in peripheral blood (0.19 x109/l [0.16 – 0.29] and 0.08 x109/l [0.05 – 0.12], respectively, p=0.004).Conclusion: Within our study we were able to detect MP in bone marrow blood. When comparing MP from bone marrow and peripheral blood, major differences with regard to cell source and concentration was present. Our data suggest that the blood – bone marrow barrier is not only existentant for cells, but also for circulating MP. DisclosuresNo relevant conflicts of interest to declare.

LVAD-Associated Coagulopathy: Contribution of Phosphatidylserine (PS+) Cellular Microparticles to the Risk of Hemorrhage and Thrombosis in Patients with Non-Pulsatile Left Ventricular Assist Devices

Purpose Left ventricular assist devices (LVADs) are associated with significant hemorrhagic and thrombotic complications, but the underlying mechanisms remain elusive. We examined whether cellular derived microparticles may be associated to adverse events in patients with LVADs. Methods and Materials We prospectively enrolled 15 patients undergoing elective implantation of a HeartMate II LVAD. Extensive coagulation profile including phosphatidylserine (PS+) cellular microparticles derived by platelets, leukocytes, erythrocytes, and endothelial cells was performed by flow cytometry. Samples were obtained upon admission (baseline), at discharge,at 3 months after discharge, and at the time of a clinically significant event. Patients were on an oral anticoagulation regimen of aspirin, warfarin (International Normalized Ratio, 2-3), and dipyridamole. Results Three patients had adverse events.At baseline, the mean percentage of PS+ microparticles in patients was higher than that in healthy controls (2.32%±1.65 vs. 0.98%±0.66) and gradually decreased during follow-up from 2.03%±1.35 (discharge) to 1.85%±1.22 (3 months). Patients with events had significantly more PS+ microparticles than did patients without events at 3 months (3.8%±0.75 vs. 1.85%±1.22, p Conclusions PS+ microparticles were elevated in patients before LVAD implantation, which is consistent with a hypercoagulable state. Anticoagulation after LVAD implantation appeared to reduce the amount of circulating PS+ microparticles in peripheral blood. Adverse thrombotic or bleeding events appeared to be preceded by elevated PS+ microparticles. The presence of these cell-derived particles may provide a procoagulant substrate that leads to significant adverse events.

Bio‐maleimide as a generic stain for detection and quantitation of microparticles

Microparticles (MP) are small fragments of cytoplasm shed from a cell surface and their role in the pathophysiology of disease is being extensively investigated. A novel staining technique for quantifying total MP in peripheral blood was evaluated in this study. Evaluation of Bodipy-maleimide (or bio-maleimide) as a stain for quantifying total MP in peripheral blood by flow cytometry. Samples were obtained from 10 healthy donors after informed consent. Plasma was prepared by sequential centrifugation at 1500 g followed by 13,000 g and stained with Annexin V and bio-maleimide. Enumeration beads were added after 15 min of incubation with the stain and samples analyzed on a FACS Canto flow cytometer. Detection and quantification of MP by bio-maleimide staining was comparable with that by Annexin V. The total mean MP level with bio-maleimide staining was 34 +/- 19.7/microl (range of 11.6-68.1/microl) and with Annexin V staining it was 38.9 +/- 29.8/microl (range of 10.6 to 112.9/microl). There was no significant difference using a paired t-test and methods were comparable using a Bland-Altman plot. Bio-maleimide is a useful and inexpensive stain to measure total MP levels in peripheral blood by flow cytometry. This technique could be employed to study thrombotic risks in a variety of disease states.

Endothelial microparticles correlate with erectile dysfunction in diabetic men

Cell-derived microparticles are supposed to be involved in endothelial dysfunction and atherogenesis. This study aimed to evaluate circulating microparticles in diabetic subjects with erectile dysfunction (ED) and their relation with endothelial dysfunction. Thirty diabetic men with ED and 20 age-matched control subjects without ED were assessed for circulating microparticles and endothelial dysfunction. Flow cytometry was used to assess microparticles by quantification of circulating endothelial (EMP, CD31(+)/CD42b(-)) and platelet (PMP, CD31(+)/CD42b(+)) microparticles in peripheral blood. Endothelium-dependent flow-mediated dilation (FMD) was evaluated in the right brachial artery after reactive hyperemia. Compared with non-diabetic subjects, diabetic men presented significantly higher numbers of EMP (P=0.001), and reduced FMD (P=0.01), with a significant inverse correlation between the number of circulating EMP and the International Index of Erectile Function (IIEF) score (r=-0.457, P=0.01). Multivariate analysis correcting for age, anthropometric indices, glucose and lipid parameters, FMD and PMP identified EMP as the only independent predictor for IIEF score (P=0.03). EMP are elevated in impotent diabetic subjects and independently involved in the pathogenesis of ED.

Detection of microparticles in peripheral blood and its clinical significance

血液中微粒是由血液细胞、血管内皮细胞在受到刺激活化后形成的,能直接而特异地反映其来源细胞的功能状态,为揭示血液或血管性疾病的发病机制、发展趋势提供依据,也可作为鉴别诊断、临床疗效的指征之一,因而检测血液中微粒具有重要意义.本文就已有的一些检测方法及其与临床疾病的相关性进行阐述,以期为深入研究提供基础与策略。

Circulating CD31 + /Annexin V + Apoptotic Microparticles Correlate With Coronary Endothelial Function in Patients With Coronary Artery Disease

Endothelial dysfunction predicts morbidity and mortality in patients at cardiovascular risk. Endothelial function may be decisively influenced by the degree of endothelial cell apoptosis. To test this hypothesis in humans, endothelial-dependent vasodilatation was invasively assessed in 50 patients with coronary artery disease (CAD) by quantitative coronary angiography during intracoronary acetylcholine infusion. Flow cytometry was used to assess endothelial cell apoptosis by quantification of circulating CD31+/annexin V+ apoptotic microparticles in peripheral blood. Increased apoptotic microparticle counts positively correlated with impairment of coronary endothelial function. Multivariate analysis revealed that increased apoptotic microparticle counts predict severe endothelial dysfunction independent of classical risk factors, such as hypertension, hypercholesterolemia, smoking, diabetes, age, or sex. In patients with CAD, endothelial-dependent vasodilatation closely relies on the degree of endothelial cell apoptosis, which is readily measurable by circulating CD31+/annexin V+ apoptotic microparticles. These findings possibly provide new options for risk assessment and may have implications for future treatment strategies of CAD.