Assessment of Physico-Chemical and Toxicological Properties of Commercial 2D Boron Nitride Nanopowder and Nanoplatelets.

Boron nitride (BN) nanomaterials have been increasingly explored for potential applications in chemistry and biology fields (e.g., biomedical, pharmaceutical, and energy industries) due to their unique physico-chemical properties. However, their safe utilization requires a profound knowledge on their potential toxicological and environmental impact. To date, BN nanoparticles have been considered to have a high biocompatibility degree, but in some cases, contradictory results on their potential toxicity have been reported. Therefore, in the present study, we assessed two commercial 2D BN samples, namely BN-nanopowder (BN-PW) and BN-nanoplatelet (BN-PL), with the objective to identify whether distinct physico-chemical features may have an influence on the biological responses of exposed cellular models. Morphological, structural, and composition analyses showed that the most remarkable difference between both commercial samples was the diameter of their disk-like shape, which was of 200-300 nm for BN-PL and 100-150 nm for BN-PW. Their potential toxicity was investigated using adenocarcinomic human alveolar basal epithelial cells (A549 cells) and the unicellular fungus Saccharomycescerevisiae, as human and environmental eukaryotic models respectively, employing in vitro assays. In both cases, cellular viability assays and reactive oxygen species (ROS) determinations where performed. The impact of the selected nanomaterials in the viability of both unicellular models was very low, with only a slight reduction of S. cerevisiae colony forming units being observed after a long exposure period (24 h) to high concentrations (800 mg/L) of both nanomaterials. Similarly, BN-PW and BN-PL showed a low capacity to induce the formation of reactive oxygen species in the studied conditions. Even at the highest concentration and exposure times, no major cytotoxicity indicators were observed in human cells and yeast. The results obtained in the present study provide novel insights into the safety of 2D BN nanomaterials, indicating no significant differences in the toxicological potential of similar commercial products with a distinct lateral size, which showed to be safe products in the concentrations and exposure conditions tested.

Assessment of Epicardial Adipose Tissue Thickness in Children with Familial Mediterranean Fever.

BACKGROUND: Familial Mediterranean fever (FMF) is suggested to be associated with increased risk of atherosclerosis. Epicardial adipose tissue (EAT) thickness is used in prediction of atherosclerotic risk. The aim of our study was to evaluate EAT thickness in FMF patients for early detection of risk of atherosclerosis and to be compared with its level in healthy controls. METHODS: Thirty 6- to 18-year-old children with FMF and 30 age- and sex-matched children (control group) were included in the study. Disease characteristics, disease severity and Mediterranean fever gene mutations were recorded. EAT thicknesses was measured by echocardiography. RESULTS: EAT in patients' group was significantly greater than that of controls (5.21 ± 2.3 vs. 2.81 ± 2.96 mm, p = 0.001) and was correlated with cholesterol level and platelets count (p = 0.047 and 0.018, respectively). CONCLUSION: This study concluded that EAT thickness was statistically increased in FMF patients than controls with a positive correlation with cholesterol level and platelet count. This finding suggests a higher risk for atherosclerosis in these patients. Follow-up study is needed to verify the effect of treatment of FMF on the EAT thickness. Further studies with larger number of patients following-up EAT are needed to verify this finding.

Flow Cytometry Assessment of Procoagulant Platelets Using a Dithiol-Reactive Probe.

Flow cytometry assessment of platelets using the combination of GSAO [4-(N-(S-glutathionylacetyl)amino)phenylarsonous acid], a dithiol-reactive probe, and P-selectin, a platelet activation marker, is a novel and powerful assay in the identification and quantification of the procoagulant subpopulation of platelets that has the capacity to support thrombin generation. In this chapter, we provide the flow cytometry protocols aimed at the study of procoagulant platelets under resting and agonist-stimulated conditions in whole blood and washed platelets of both human and murine (mouse) samples.

Assessment of bone healing ability of calcium phosphate cements loaded with platelet lysate in rat calvarial defects.

Injectable calcium phosphate cements have been used as a valid alternative to autologous bone grafts for bone augmentation with the additional advantage of enabling minimally invasive implantation procedures and for perfectly fitting the tissue defect. Nevertheless, they have low biodegradability and lack adequate biochemical signaling to promote bone healing and remodeling. In previous in vitro studies, we observed that the incorporation of platelet lysate directly into the cement paste or loaded in hyaluronic acid microspheres allowed to modulate the cement degradation and the in vitro expression of osteogenic markers in seeded human adipose derived stem cells. The present study aimed at investigating the possible effect of this system in new bone formation when implanted in calvarial bilateral defects in rats. Different formulations were assessed, namely plain calcium phosphate cements, calcium phosphate cements loaded with human platelet lysate, hybrid injectable formulations composed of the calcium phosphate cement incorporating hyaluronin acid non-loaded microparticles (20% hyaluronin acid) or with particles loaded with platelet lysate. The degradability and new bone regrowth were evaluated in terms of mineral volume in the defect, measured by micro-computed tomography and histomorphometric analysis upon 4, 8 and 12 weeks of implantation. We observed that the incorporation of hyaluronin acid microspheres induced an overly rapid cement degradation, impairing the osteoconductive properties of the cement composites. Moreover, the incorporation of platelet lysate induced higher bone healing than the materials without platelet lysate, up to four weeks after surgery. Nevertheless, this effect was not found to be significant when compared to the one observed in the sham-treated group.

Assessment of nucleic acid modification induced by amotosalen and ultraviolet A light treatment of platelets and plasma using real-time polymerase chain reaction amplification of variable length fragments of mitochondrial DNA.

BACKGROUND: Pathogen inactivation methods are increasingly used to reduce the risk of infections after transfusion of blood products. Photochemical treatment (PCT) of platelets (PLTs) and plasma with amotosalen and ultraviolet A (UVA) light inactivates pathogens and white blood cells through formation of adducts between amotosalen and nucleic acid that block replication, transcription, and translation. The same adducts block the amplification of nucleic acids using polymerase chain reaction (PCR) in a manner that correlates with the number of adducts formed, providing a direct quality control (QC). Current QC measures for PCT rely on indirect methods that measure the delivered UVA dose or percent residual amotosalen after illumination, rather than directly measuring nucleic acid modification. STUDY DESIGN AND METHODS: Endogenous mitochondrial DNA (mtDNA), which is detectable in PLT and plasma units, was chosen as a target for the quantification of photochemically induced modifications. DNA was extracted from untreated or amotosalen and UVA-treated PLTs or plasma, and mtDNA fragments of variable lengths were quantified using a real-time PCR inhibition assay. RESULTS: PCT induced increasing real-time PCR inhibition of mtDNA amplification for larger amplicon sizes. Amplification was unaffected by treatment with amotosalen or UVA alone, whereas up to 3 log inhibition was observed after PCT. Blinded PCR testing of a panel of 110 samples each, from PLT or plasma components prepared for routine use within a blood center, allowed 100% discrimination between untreated and treated units. CONCLUSION: Our initial findings indicate that an adequately sensitive, quantitative real-time PCR inhibition assay targeting mtDNA could provide a valuable tool to confirm and monitor PCT.

Stability validation of paraformaldehyde-fixed samples for the assessment of the platelet PECAM-1, P-selectin, and PAR-1 thrombin receptor by flow cytometry.

Sample fixation for storage and/or transportation represents an unsolved challenge for multicenter clinical trials assessing serial changes in platelet activity, or monitoring various antiplatelet regimens. Whole blood flow cytometry represents a major advance in defining platelet function, although special training and expensive equipment is required. We sought to determine how fixation with 2% paraformaldehyde (PFA), and storage of blood samples over 1 week affects the flow cytometry readings for both intact and thrombin-activating four major surface platelet receptors. Whole blood platelet expression of PECAM-1, P-selectin, PAR-1 inactive receptor (SPAN-12), and cleaved (WEDE-15) epitope was assessed immediately after blood draw, after staining with 2% PFA, and at day 1, 3, 5, and 7. The study was performed in 6 volunteers with multiple risk factors for vascular disease, not receiving any antiplatelet agents. Staining with PFA resulted in a slight decrease of fluorescence intensity, especially for PECAM-1, while antigen expression at day 1, 3 and 5 remains consistent, and highly reproducible. At day 7 there was a small but inconsistent trend towards diminished fluorescence intensity. The platelet data were consistent while validated with the isotype-matched irrelevant antibody. These data suggest that there is a 5 day window to perform final flow cytometry readings of whole blood PFA-fixed inactivated platelet samples. In contrast, thrombin activation cause gradual loss of flow cytometry signal, and cannot be recommended for long-term storage. This is critical logistic information for conducting multicenter platelet substudies within the framework of major clinical trials.

Assessment of platelet growth factors in supernatants from rehydrated freeze-dried equine platelets and their effects on fibroblasts in vitro.

OBJECTIVE: To determine whether platelet growth factors are preserved in supernatants obtained from rehydrated trehalose-stabilized, freeze-dried (lyophilized) equine platelets and whether those growth factors stimulate fibroblast proliferation and migration and enhance fibroblast-associated contraction in a collagen gel assay. ANIMALS: 6 clinically normal adult horses. PROCEDURES: Blood samples were obtained from 6 horses, and washed platelets were prepared via differential centrifugation. Washed platelets were freeze-dried in a physiologic buffer with a mixture of trehalose and polyethylene glycol 4000. Rehydrated platelet supernatants and releasates prepared from fresh washed platelets stimulated with thrombin or platelet-activating factor were evaluated for transforming growth factor beta1 and platelet-derived growth factor-BB by use of ELISAs. Effects of rehydrated freeze-dried platelet supernatants on fibroblast proliferation, migration, and collagen gel contraction were compared with effects of 1%, 2.5%, or 10% fetal bovine serum (FBS). RESULTS: Supernatants from freeze-dried platelets contained similar amounts of growth factors as thrombin- and platelet-activating factor-stimulated platelet releasates. The supernatants significantly enhanced fibroblast proliferation and migration in a scratch assay, compared with FBS-free control or low (1%) FBS conditions. Additionally, supernatants from freeze-dried platelets enhanced contraction of fibroblast-seeded collagen gels, compared with the effect of 1% FBS. CONCLUSIONS AND CLINICAL RELEVANCE: The preparation technique preserved platelet growth factors, enhanced fibroblast proliferation and migration, and improved fibroblastseeded collagen gel contraction under conditions of low FBS concentration; these platelet supernatant preparations may prove useful as an aid to conventional wound management.

Ethylenediaminetetraacetic acid plus citrate-theophylline-adenosine-dipyridamole (EDTA-CTAD): a novel anticoagulant for the flow cytometric assessment of platelet and neutrophil activation ex vivo in whole blood.

Ethylenediaminetetraacetic acid (EDTA) is the anticoagulant recommended for full blood counts, citrate is recommended for coagulation and platelet studies, and citrate-theophylline-adenosine-dipyridamole (CTAD) inhibits platelet activation. Because the combination of EDTA and CTAD (E/C) is better than EDTA or CTAD alone for measuring platelet parameters on the ADVIA 120 Haematology System, we investigated whether it also offers advantages for the flow cytometric assessment of platelet and/or neutrophil activation and platelet-leucocyte aggregate formation ex vivo. Blood from healthy subjects was collected into E/C or citrate, kept at room temperature or at 4 degrees C, and analysed 0 to 360 min later in the ADVIA 120 and by immunofluorescent flow cytometry. Platelet count, mean platelet volume, number of platelet clumps, mean platelet component, numbers of CD62P(+) platelets and platelet-leucocyte aggregates, and expression of CD11b on neutrophils changed little over 360 min in blood with E/C kept at 4 degrees C. In contrast, one or more parameter changed when blood was kept with E/C at ambient temperature or with citrate at either temperature. The use of E/C in in vitro and in vivo studies is illustrated. Platelet and neutrophil activation status ex vivo can be reliably assessed if blood is collected into E/C, held at 4 degrees C, and analysed within 6 h.

Failure of platelet parameters and biomarkers to correlate platelet function to severity and etiology of heart failure in patients enrolled in the EPCOT trial. With special reference to the Hemodyne hemostatic analyzer. Whole Blood Impedance Aggregometry for the Assessment of Platelet Function in Patients with Congestive Heart Failure.

Data from small studies have suggested the presence of platelet abnormalities in patients with congestive heart failure (CHF). We sought to characterize the diagnostic utility of different platelet parameters and platelet-endothelial biomarkers in a random outpatient CHF population investigated in the EPCOT ('Whole Blood Impedance Aggregometry for the Assessment of Platelet Function in Patients with Congestive Heart Failure') Trial. Blood samples were obtained for measurement of platelet contractile force (PCF), whole blood aggregation, shear-induced closure time, expression of glycoprotein (GP) IIb/IIIa, and P-selectin in 100 consecutive patients with CHF. Substantial interindividual variability of platelet characteristics exists in patients with CHF. There were no statistically significant differences when patients were grouped according to incidence of vascular events, emergency revascularization needs, survival, or etiology of heart failure. Aspirin use did not affect instrument readings either. PCF correlates very poorly with whole blood aggregometry (r(2) = 0.023), closure time (r(2) = 0.028), platelet GP IIb/IIIa (r(2) = 0.0028), and P-selectin (r(2) = 0.002) expression. Furthermore, there was no correlation with brain natriuretic peptide concentrations, a marker of severity and prognosis in heart failure reflecting the neurohumoral status. Patients with heart failure enrolled in the EPCOT Trial exhibited a marginal, sometimes oppositely directed change in platelet function, challenging the diagnostic utility of these platelet parameters and biomarkers to serve as useful tools for the identification of platelet abnormalities, for predicting clinical outcomes, or for monitoring antiplatelet strategies in this population. The usefulness of these measurements for assessing platelets in the different clinical settings remains to be explored. Taken together, opposite to our expectations, major clinical characteristics of heart failure did not correlate well with the platelet characteristics investigated in this study.

Assessment of in vitro-generated platelet microparticles using a modified flow cytometric strategy.

Quantification of platelet microparticles (PMPs) may be a useful marker for the detection of in vivo platelet activation. Optimisation of flow cytometric methods for detection and quantification of PMPs has not been systemically evaluated. This study reports the optimisation of flow cytometric procedures for the detection of PMPs, the determination of limits of size detection using microbeads, and the characterisation of PMP generation by in vitro activation of platelets using collagen and adenosine 5' diphosphate (ADP). Fluorescent and plain microbeads proved useful for defining the limits of the flow cytometer in detecting PMPs. A systematic calibration of the forward scatter (FS) threshold parameter (size) of the flow cytometer using microbeads allowed for the detection of very small particles (down to 0.1 microm diameter). PMPs generated in vitro using ADP and collagen were reliably detected by flow cytometry using monoclonal antibodies (MAb) directed towards platelet surface membrane glycoproteins (Gp). The PMP events were detected in the FS low (i.e., small size events) and fluorescence (FL) high (i.e., platelet Gp MAb-labelled events) region. PMPs of different size profiles were observed for each of the agonists. Flow cytometry can be used as a tool in the assessment of PMPs. As detection of particles of this type is at the limit of resolution of flow cytometers, careful attention is required with the choice of platelet-specific MAb, isotype control, and optimisation of procedure setup and performance.

Reticulated platelet counts in the assessment of thrombocytopenic disorders.

Reticulated platelets (RP) are the youngest platelets in the circulation and can be measured by analysing the RNA content of platelets from whole blood or platelet-rich plasma by flow cytometry. Increased RP are indicative of increased production of platelets. Despite the current lack of standardization for the measurement of RP, it is useful in the assessment of patients with ITP by aiding the distinction of these patients from those with decreased platelet production. RP counts also have a role in the assessment of the complicated patient with multiple possible aetiologies for thrombocytopenia. Measurement of the RP count may hold predictive value for marrow recovery following myelosuppressive or myeloablative chemotherapy, and may play a role in monitoring the administration of the various thrombopoietins currently under clinical trial.

Assessment of disease activity in ulcerative colitis by faecal calprotectin, a novel granulocyte marker protein.

This study comprised 62 outpatients with ulcerative colitis who underwent 64 colonoscopies. The disease activity was evaluated according to endoscopic and histological criteria. The results revealed a significant correlation between both the endoscopic as well as the histological gradings of disease activity and faecal calprotectin. The median faecal calprotectin levels in the control group (6 mg/l) and in the patients with no or low disease activity (11.5 mg/l) were significantly different (p < 0.0001). The median calprotectin level among patients with active disease was 68 mg/l which was significantly different from the latter group (p < 0.0001). Furthermore, we suggest that the degree of inflammation rather than the extent of the disease determined the faecal calprotectin levels. In conclusion, assessment of faecal calprotectin seems to be a marker of disease activity in patients with ulcerative colitis.

Assessment of platelet cytosolic concentration of free magnesium in healthy subjects.

We measured basal cytosolic concentration of free Mg2+ ([Mg2+]i) in platelets from 30 healthy volunteers by using mag-fura-2, a new fluorescent Mg2+ indicator. The mean [Mg2+]i was 381 +/- 22 mumol/L, with values ranging from 226 to 771 mumol/L. The day-to-day intrasubject coefficient of variation was relatively small (3.6%). [Mg2+]i was significantly higher in men than in women (430 +/- 38 vs 332 +/- 17 mumol/L, p < 0.03), and was not correlated with age. There was no significant relation between platelet [Mg2+]i and serum total Mg concentration. The results indicate that the gender but not age may decide intracellular Mg2+ status. In addition, the mechanisms that regulate [Mg2+]i may be independent of those that influence the extracellular concentration of Mg.

Assessment of vitamin E nutritional status in sheep.

The effects of supplemental dietary vitamin E (as DL-alpha-tocopheryl acetate; 0, 15, 30, and 60 IU/d) on serum, platelet, and muscle tocopherol and lipid (cholesterol and triglycerides) concentrations in 32 sheep were investigated in a 60-d trial. Serum, platelet, and muscle alpha-tocopherol concentrations increased linearly (P < .05) with treatment. Platelet tocopherol concentrations were more sensitive to vitamin E intake than either serum or muscle tocopherol. There were no effects on serum lipid concentrations. There were low correlations (P > .05) between serum or platelet tocopherol and either cholesterol or triglycerides or the sum of the two lipid fractions. Correlations between serum or platelet tocopherol and muscle tocopherol were also low (P > .05). Although platelet tocopherol was more sensitive to vitamin E intake than serum tocopherol, serum tocopherol concentrations can be reliably used to estimate vitamin E status. Expressing serum tocopherol relative to blood lipids did not improve the relationship between serum tocopherol and vitamin E intake.

Glycoproteins Ib and IIb/IIIa in the quality assessment of platelet concentrates during storage.

We have previously shown that levels of soluble glycocalicin (GC) in plasma supernatants derived from units of platelet concentrates (PC) increase progressively during storage. We now report further studies which show that the levels of both microparticle-bound and soluble GC in PC during storage are influenced by exposure of PC samples to EDTA and treatment of PC packs with ultraviolet B (UVB) irradiation. EDTA leads to a significant increase in the release of microvesicle-bound and soluble GC, while UVB irradiation leads to a dose- and rate-dependent increase in GC release. Paradoxically, UVB leads to an unexpected decrease in supernatant levels of von Willebrand factor (vWf) during storage which contrasts with its increase in untreated, stored PC. Moreover, an increase in GC release during storage is associated with a corresponding decrease in platelet size as determined by measurement of mean platelet volume (MPV) in citrated PC. The GC release is significantly correlated with standard platelet functional tests and other new generation tests such as dMPV and supernatant levels of vWf. In addition, preliminary results show the presence of microparticle-bound and soluble glycoprotein (Gp) IIb/IIIa in the supernatant plasma of stored PC. Our results suggest that supernatant levels of GpIb, GpIIb/IIIa, and vWf, together with alteration in MPV, provide essential new informative parameters for quality assessment of PC.

Erythrocytes, erythrocyte membranes, neutrophils and platelets as biopsy materials for the assessment of zinc status in humans.

During a controlled zinc depletion-repletion study, fifteen men aged 25.3 (SD 3.3) years were fed on a low-Zn diet with high phytate:Zn and phytate x calcium:Zn molar ratios for 7 weeks, followed by a 2 week repletion period when 30 mg supplemental Zn/d was given. Changes in plasma, urine, and hair Zn concentrations, taste acuity, and cellular immune response confirmed the development of mild Zn deficiency. Zn concentrations in neutrophils, platelets, erythrocytes and erythrocyte membranes, mean platelet volume, and activities of alkaline phosphatase (EC 3.1.3.1) and alpha-D-mannosidase (EC 3.2.1.24) in neutrophils did not respond to changes in Zn status. In contrast, alkaline phosphatase activity in erythrocyte membranes showed a significant decline which was consistent in all subjects (nmol product formed/min per mg protein; baseline v. 7-week Zn depletion, 0.656 (SD 0.279) v. 0.506 (SD 0.230), at 7 weeks; P < 0.05); neutral phosphatase activity remained unchanged. Alkaline phosphatase activity in erythrocyte membranes may be a potential index of Zn status in humans.

Flow cytometric assessment of platelet function in patients with peripheral arterial occlusive disease.

This study compared new and traditional measures of platelet function in 16 patients with severe peripheral arterial occlusive disease and 15 age-matched controls. Circulating platelets were characterized by the use of fluorescence flow cytometry to assess platelet aggregate formation and expression of the secretion-dependent alpha granule membrane protein GMP-140, by measurement of plasma beta-thromboglobulin (beta-TG), and by performance of platelet-rich plasma aggregation studies. In addition, blood samples were treated with graded concentrations of adenosine diphosphate (ADP; 0 to 10 mumols/L) to characterize by fluorescence flow cytometry the secretory and aggregatory responses to mild stimulation. No differences were detected between the two groups with regard to platelet function in unstimulated circulating blood by use of these techniques. Values (mean +/- SEM) observed were: GMP-140-positive platelets, 11% +/- 3% versus 13% +/- 2%; platelet aggregates in circulating whole blood, 4% +/- 1% versus 9% +/- 3%; plasma beta-TG, 92 +/- 12 versus 94 +/- 22 ng/ml; and ED50 (concentration of ADP required to produce half maximal aggregation), 3.8 +/- 1.1 versus 3.1 +/- 0.5 mumol/L in the patients with peripheral arterial occlusive disease and controls, respectively. Treatment with ADP caused a dose-related increase in GMP-140 expression in both groups, without significant differences in this parameter between the groups at any given concentration. However, stimulation with ADP concentrations greater than 1 mumol/L resulted in more frequent aggregate formation in the control than in the peripheral arterial occlusive disease group (25% +/- 4% versus 11% +/- 2%, respectively at 5.0 mumols/L, p = 0.002).(ABSTRACT TRUNCATED AT 250 WORDS)