DTT Assay Research Articles

Gaseous and aerosol emissions from open burning of tree pruning and hedge trimming residues: Detailed composition and toxicity

Gaseous and PM10 samples were collected during the open burning of pruning residues (olive branches and garden waste) and characterised by distinct analytical techniques to obtain comprehensive chemical emission profiles. Oxidative potential (dithiothreoitol and ascorbic acid assays) and cell viability tests were also performed with the aim of evaluating aerosol toxicity. Emission factors (EFs) were as follows (g kg−1 biofuel, dry basis): 1537–1672 for CO2, 41.9–80 for CO, 2.74–6.6 for CH4, 0.89–3.51 for ethane, 0.79–1.78 for ethylene and 0.56–3.47 for formaldehyde. Emissions of PM10, organic carbon (OC) and elemental carbon (EC) were in the ranges 8–41, 3–18, and 0.4–1.5 g kg−1 biofuel, dry basis, respectively. OC accounted for 35–45% of the total PM10 mass, while EC contributed between around 3% and 5%. WSOC/OC ratios varied from 0.4 to 0.6, revealing that a substantial portion of the carbon emitted was hydrosoluble. Water soluble ions constituted around 8–21% of the PM10 mass, with potassium and chloride as the most abundant ions in all samples. Levoglucosan, widely used a reliable biomass burning tracer, was found in significant amounts in all samples (up to 1.2% of the PM10 mass). Retene, generally pointed out as a biomass combustion biomarker, was the predominant PAH. WSOC and some PAHs showed significant positive correlations with the intrinsic OP measured with the DTT assay, while the OPAA was significantly correlated with some trace metals, such as Fe or Ni. All samples significantly reduced the viability of alveolar epithelial cells.

Discovery of a Highly Promising Disulfide Derivative Scaffold as Inhibitor of SARS-CoV-2 Main Protease.

The main protease (Mpro) of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2) represents a promising target for antiviral drugs aimed at combating COVID-19. Consequently, the development of Mpro inhibitor is an ideal strategy for combating the virus. In this study, we identified twenty-two dithiocarbamates (1 a-h), dithiocarbamate-Cu(II) complexes (2 a-hCu) and disulfide derivatives (2 a-e, 2 i) as potent inhibitors of Mpro, with IC50 value range of 0.09-0.72, 0.9-24.7, and 15.1-111 μM, respectively, through FRET screening. The enzyme kinetics, inhibition mode, jump dilution, and DTT assay revealed that 1 g may be a partial reversible inhibitor, while 2 d and 2 f-Cu are the irreversible and dose- and time-dependent inhibitors, potentially covalently binding to the target. Binding of 2 d, 2 f-Cu, and 1 g to Mpro was found to decrease the stability of the protein. Additionally, DTT assays and thermal shift assays indicated that 2 f-Cu and 2 d are the nonspecific and promiscuous cysteine protease inhibitor. ICP-MS implied that the inhibitory activity of 2 f-Cu may stem from the uptake of Cu(II) by the enzyme. Cytotoxicity assays demonstrated that 2 d and 1 g exhibit low cytotoxicity, whereas 2 f-Cu show certain cytotoxicity in L929 cells. Overall, this work presents two promising scaffolds for the development of Mpro inhibitors to combat COVID-19.

Spatially resolved chemical data for PM10 and oxidative potential source apportionment in urban-industrial settings

This study proposes the use of spatially resolved data to identify local sources contributing to PM10 mass concentration and OP in urban-industrial contexts. Over a 18-month monitoring campaign spanning from October 2020 to May 2022, a low-volume PM10 sampling device was employed at 12 sites in the Sacco River Valley, Italy. PM10 samples were analyzed for OP by applying the DTT, AA, and DCFH assays, and for 77 chemical parameters: the main chemical components (major elements, inorganic ions, EC, OC, levoglucosan) and the water-soluble and insoluble fractions of 28 elements. Source apportionment via PMF analysis on spatially resolved data revealed seven PM10 source contributions, notably biomass burning and soil dust. Biomass burning predominated in winter, while soil dust prevailed in summer. Spatial variability in source contributions was observed due to diverse biomass types, domestic heating, and proximity to the cement plant and transportation networks. Bubble maps and FPCA represented OP and PM10 source relationships, highlighting biomass burning as the main contributor to OPDTT and OPDCFH, while non-exhaust traffic contributed significantly to OPAA. Findings from this study provide valuable insights into planning effective air pollution mitigation policies aimed at enhancing air quality in urban-industrial contexts.

The oxidative potential of fine ambient particulate matter in Xinxiang, North China: Pollution characteristics, source identification and regional transport

Atmospheric fine particulate matter (PM2.5) can trigger the production of cytotoxic reactive oxygen species (ROS), which can trigger or exacerbate oxidative stress and pulmonary inflammation. We collected 111 daily (∼24 h) ambient PM2.5 samples within an urban region of North China during four seasons of 2019–2020. PM2.5 samples were examined for carbonaceous components, water–soluble ions, and elements, together with their oxidative potential (represent ROS–producing ability) by DTT assay. The seasonal peak DTTv was recorded in winter (2.86 ± 1.26 nmol min−1 m−3), whereas the DTTm was the highest in summer (40.6 ± 8.7 pmol min−1 μg−1). WSOC displayed the highest correlation with DTT activity (r = 0.84, p < 0.0001), but the influence of WSOC on the elevation of DTTv was extremely negligible. Combustion source exhibited the most significant and robust correlation with the elevation of DTTv according to the linear mixed–effects model result. Source identification investigation using positive matrix factorization displayed that combustion source (36.2%), traffic source (30.7%), secondary aerosol (15.7%), and dust (14.1%) were driving the DTTv, which were similar to the results from the multiple linear regression (MLR) analysis. Backward trajectory analysis revealed that the major air masses originate from local and regional transportation, but PM2.5 OP was more susceptible to the influence of short–distance transport clusters. Discerning the influence of chemicals on health–pertinent attributes of PM2.5, such as OP, could facilitate a deep understanding of the cause–and–effect relationship between PM2.5 and impacts.

Bivalirudin Monitoring in Pediatric Ventricular Assist Device and Extracorporeal Membrane Oxygenation: Analysis of Single-Center Retrospective Cohort Data 2018-2022.

The activated partial thromboplastin time (aPTT) is the most frequently used monitoring assay for bivalirudin in children and young adults on mechanical circulatory support including ventricular assist devices (VADs) and extracorporeal membrane oxygenation (ECMO). However, intrinsic variability of the aPTT complicates management and risks bleeding or thrombotic complications. We evaluated the utility and reliability of a bivalirudin-calibrated dilute thrombin time (Bival dTT) assay for bivalirudin monitoring in this population. Retrospective analysis of clinical data (including aPTT, dilute thrombin time [dTT]) and results of residual plasma samples from VAD patients were assessed in two drug-calibrated experimental assays. One assay (Bival dTT) was validated for clinical use in VAD patients, and subsequently used by clinicians in ECMO patients. Pearson correlation and simple linear regression were used to determine R2 correlation coefficients between the different laboratory parameters using Statistical Package for Social Sciences (Armonk, NY). ICUs at Cincinnati Children's Hospital Medical Center. Children on VAD or ECMO support anticoagulated with bivalirudin. None. One hundred fifteen plasma samples from 11 VAD patients were analyzed. Both drug-calibrated experimental assays (anti-IIa and Bival dTT) showed excellent correlation with each other ( R2 = 0.94) and with the dTT ( R2 = 0.87), but poor correlation with aPTT ( R2 = 0.1). Bival dTT was selected for validation in VAD patients. Subsequently, clinically ordered results (105) from 11 ECMO patients demonstrated excellent correlation between the Bival dTT and the standard dTT ( R2 = 0.86) but very poor correlation with aPTT ( R2 = 0.004). APTT is unreliable and correlates poorly with bivalirudin's anticoagulant effect in ECMO and VAD patients. A drug-calibrated Bival dTT offers superior reliability and opportunity to standardize results across institutions. Additional studies are needed to determine an appropriate therapeutic range and correlation with clinical outcomes.

Physicochemical Characterization and Oxidative Potential of Iron-Containing Particles Emitted from Xuanwei Coal Combustion.

Respiratory diseases have been proven to be directly related to air pollutants. Xuanwei, located in South China, has been known to have the highest mortality rate for lung cancer in China because of the air pollutants emitted through local coal combustion. However, the mechanism of lung cancer induced by air pollutants is not clear. Based on the fact that a large number of iron-bearing mineral particles was found in Xuanwei coal combustion particles, the iron-containing particles were hypothesized to play important roles in the pathogenesis of the high incidence rate of lung cancer in this area. In this study, raw coal samples were collected from a coal mine in the Xuanwei area. Size-resolved particles emitted from the raw coal samples were collected using an Anderson high-volume sampler. Mineralogical characterization and an assessment of the oxidative potential of the iron-containing particles were conducted using cutting-edge technologies, and the biological activity of the particles were evaluated via DTT assay. Our data showed that the iron-containing minerals accounted for more than 10% of the measured particles emitted from Xuanwei coal combustion samples. The content analysis of ·OH generated from Xuanwei coal combustion particles showed that ·OH content was dependent on the size of particles in the surrogated lung fluid. The concentration of ·OH increased as the particle size decreased. The DTT assay data further demonstrated that when the mass concentration of dissolved irons increased, the oxidation potential of the particles increased. The highest proportion of divalent iron in the total dissolved iron was found in the submicron particles in low pH solution(pH = 1), which indicated that the oxidative potential induced by submicron particles was stronger than that induced by coarse particles and fine particles. Armed with the above data, the toxicological mechanism of the iron-containing mineral particles can be investigated further.

Exposure to urban nanoparticles at low PM_1 concentrations as a source of oxidative stress and inflammation

Exposures to fine particulate matter (PM_1) have been associated with health impacts, but the understanding of the PM_1 concentration-response (PM_1-CR) relationships, especially at low PM_1, remains incomplete. Here, we present novel data using a methodology to mimic lung exposure to ambient air (2<PM_1< 60 upmug m^{-3}), with minimized sampling artifacts for nanoparticles. A reference model (Air Liquid Interface cultures of human bronchial epithelial cells, BEAS-2B) was used for aerosol exposure. Non-linearities observed in PM_1-CR curves are interpreted as a result of the interplay between the aerosol total oxidative potential (OP_t) and its distribution across particle size (d_p). A d_p-dependent condensation sink (CS) is assessed together with the distribution with d_p of reactive species . Urban ambient aerosol high in OP_t, as indicated by the DTT assay, with (possibly copper-containing) nanoparticles, shows higher pro-inflammatory and oxidative responses, this occurring at lower PM_1 concentrations (< 5 upmug m^{-3}). Among the implications of this work, there are recommendations for global efforts to go toward the refinement of actual air quality standards with metrics considering the distribution of OP_t with d_p also at relatively low PM_1.

Chromogenic and Clot-Based Bivalirudin Assays for Monitoring Anticoagulation.

Direct thrombin inhibitors (DTIs) are usually monitored with the activated partial thromboplastin time (aPTT) or activated clotting time (ACT). Both are complex assays with multiple enzymatic steps, and performance may be influenced by physiologic and pathologic factors unrelated to the DTI. Simpler systems, such as clot-based dilute thrombin time (dTT) and chromogenic anti-factor IIa assays, have been developed for monitoring DTIs, but there is limited data on their performance in clinical settings. Medical records of patients who received bivalirudin between March 2020 and April 2022 at a single institution were reviewed for demographic data and adverse outcomes. Plasma samples drawn for aPTT testing were analyzed with chromogenic anti-IIa and dTT bivalirudin assays. Results were compared to bivalirudin dosing. Results of aPTT assays from 32 patients were compared with the chromogenic (n = 136) and dTT (n = 120) bivalirudin assays. Correlations between the aPTT and the chromogenic and dTT assays were poor (Spearman coefficients 0.55 and 0.62, respectively). There was a stronger correlation when results of the chromogenic and dTT assays were compared to each other (Spearman coefficient 0.92). When assay results were compared to bivalirudin dose, there were stronger correlations with the chromogenic and dTT assays than with the aPTT (Spearman coefficients 0.51, 0.63 and 0.22, respectively). There was considerable variation between results of specific bivalirudin assays and the aPTT. While bivalirudin assay results correlated better with administered drug dose, suggesting improving reliability, more studies are needed to determine if there is correlation between testing and clinical outcomes.

Comparisons between diluted thrombin time, ecarin chromogenic assays, and UPLC-MS for plasma level dabigatran quantification: Results from DRIVING study.

The knowledge of dabigatran levels is helpful for decision-making in specific situations such as urgent surgery or when the question of reversal arises (uncontrolled bleeding, eligibility for thrombolysis). However, a limited number of observational studies are available regarding comparisons between quantification methods. The objective of the study was to compare dabigatran plasma levels using three assays including the reference method (high-performance liquid chromatography coupled with mass spectrometry), focusing on the agreement around the 30-50 ng/mL clinically relevant thresholds. Sixty healthy volunteers from DRIVING trial (NCT01627665) were given a single 300-mg dabigatran etexilate dose. Serial blood samplings were performed at pre-defined time points (0 to 24 h). We analyzed plasma samples using ultra-performance-liquid chromatography coupled with tandem mass spectrometry (UPLC-MS) (dabigatran reference method); ii/diluted thrombin time (dTT) (Hemoclot-DTI-Hyphen-Biomed); iii/ecarin-based chromogenic assay (ECA-II-Stago). Nine hundred sixty samples were analyzed using the three assays (2759 values). dTT and ECA-II values were highly correlated with those of UPLC-MS (Deming regression). Most values >50 ng/mL were higher using dTT and ECA-II compared to UPLC-MS: biases were constant, +14% and +16% with dTT and ECA-II, respectively (Bland-Altman plots), suggesting that active metabolites accounted for ~15% of thrombin inhibition. Regarding values <30 ng/mL, 30-50 ng/mL, or ≥50 ng/mL, the agreement probability between dTT and ECA-II was of 90.6% [88.4-92.5] (Cohen's kappa coefficient 0.84). dTT and ECA-II assays rapidly provide accurate dabigatran-level results for clinical practice, both assays being suitable in emergency, taking into account the thrombin inhibitory effect of dabigatran metabolites.

Use of a Calibrated, Drug-Specific DTT Assay to Improve Bivalirudin Monitoring in Pediatric Extracorporeal Membrane Oxygenation (ECMO)

Background: Balancing the hemostatic system in anticoagulated ECMO patients to optimally prevent thrombosis and bleeding is challenging. Although the traditional anticoagulant of choice for ECMO has been unfractionated heparin (UFH), use of the direct thrombin inhibitor bivalirudin is growing, due to reported reductions in bleeding and thrombotic complications. Bivalirudin monitoring remains problematic, as the most widely available and commonly used assay, the activated partial thromboplastin time (aPTT), correlates poorly with other assays of bivalirudin effect. An accurate, standardized monitoring test is needed. In a prior study we investigated use of a research-only dilute thrombin time (dTT) assay calibrated to bivalirudin concentration (Hemoclot, Hyphen-Biomed, FR) in a pediatric ventricular assist device (VAD) population. This bivalirudin dTT had excellent correlation with both the clinical dTT (R2 = 0.991) and with a research-only, drug-calibrated factor IIa chromogenic assay (BiophenDT, Hyphen-Biomed, FR) (R2 = 0.94), but poor correlation with the aPTT (R2 =0.1). This led to an institutional practice change to utilize the clinical dTT and the bivalirudin dTT assay for monitoring bivalirudin in VAD patients. Clinicians started to include these tests as well as the aPTT for patients on ECMO; however, validation of the test in the ECMO population has not been reported. Aim: The aim of this quality improvement project was to validate how the bivalirudin dTT correlated with the clinically ordered dTT (c-dTT) and aPTT in pediatric ECMO patients. Methods: A retrospective review of pediatric ECMO patients ages 0-18 years treated with bivalirudin was conducted. Bivalirudin dosing, monitoring and titration was at the discretion of the treating physician. Clinically ordered laboratory testing of interest included c-dTT, bivalirudin dTT, and aPTT. The bivalirudin dTT uses a known quantity of human thrombin added to a 1:10 diluted plasma sample to initiate clotting. Clotting time (seconds) is related to bivalirudin concentration using drug-specific controls and calibrators of low and high (1.5 and 4 microgram/mL) bivalirudin (Biophen, Hyphen-Biomed, FR). Clinical data is reported descriptively. The bivalirudin dTT was compared to aPTT and c-dTT results using Pearson correlation and an R2 coefficient determined by simple linear regression. Data analysis on clinical outcomes including circuit thrombosis, patient thrombosis, and bleeding events is ongoing. Results: In total, 124 clinically obtained, concurrent c-dTT, aPTT, and bivalirudin dTT results from 6 pediatric ECMO patients receiving bivalirudin were evaluated. Ages ranged from 0-14 years (mean 3.5 years) with 4 patients started on ECMO in the neonatal period (<1 month of age). Indications for ECMO included congenital heart disease, congenital diaphragmatic hernia, respiratory failure, and cardiogenic shock. Mean bivalirudin infusion rate was 0.6 mg/kg/hr (range 0.15-0.84mg/kg/hr). Average duration on bivalirudin was 8 days (range 4-11) for 5 of the 6 patients, with 1 patient on for > 150 days. The mean aPTT was 78.5 seconds (range 55.9-105.5 seconds). The mean c-dTT was 51 seconds (range 25.4-71.1 seconds). The bivalirudin assay had poor correlation with aPTT (R2 = 0.273, Fig 1) and excellent correlation with the c-dTT (R2 = 0.899, Fig 2). A c-dTT range of 35-65 seconds corresponded with a bivalirudin dTT level of 0.55-1.8mcg/mL. Discussion: Here we report results of the clinical use of a bivalirudin dTT monitoring assay in pediatric ECMO patients. Similar to prior results in VADs, the aPTT showed poor correlation with the bivalirudin dTT. The c-dTT had excellent correlation with the bivalirudin dTT and results were so stable for an infusion rate that clinicians ordered less frequent monitoring labs, compared to their practice for UFH. More research and outcomes data is needed to determine the appropriate therapeutic range for optimizing bivalirudin effect and minimizing complications. Validation of a bivalirudin-specific assay that can be used by many institutions will facilitate collaboration on outcomes-based research. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Monitoring Direct Thrombin Inhibitors With Calibrated Diluted Thrombin Time vs Activated Partial Thromboplastin Time in Pediatric Patients.

Activated partial thromboplastin time (aPTT) is the primary test used to monitor intravenous (IV) direct thrombin inhibitors (DTIs) but has many limitations. The plasma diluted thrombin time (dTT) has shown better correlation with DTI levels than aPTT. This study compared dose-response curves for dTT and aPTT in pediatric patients receiving argatroban and bivalirudin. A retrospective review of pediatric patients treated with argatroban (n = 45) or bivalirudin (n = 14) monitored with dTT and aPTT. The dTT assay was calibrated to report DTI concentrations in µg/mL for argatroban and bivalirudin with good analytic sensitivity and specificity. The dTT was fivefold more likely to show a stable dose-response slope than the aPTT (P < .0002; odds ratio, 4.9). For patients in whom both dTT and aPTT showed a significant correlation between dose and assay results, dTT had a higher average correlation factor compared with aPTT (P = .007). Argatroban dose-response slopes showed more inter- and intrapatient variation than bivalirudin (dose-response slope coefficient of variation, 132% vs 52%). The dTT assay was more likely to show a stable dose response and have a stronger correlation with DTI dose than aPTT. Argatroban shows more variation in dose response than bivalirudin.

Inter-annual variability of source contributions to PM10, PM2.5, and oxidative potential in an urban background site in the central mediterranean

Airborne particulate matter (PM) is studied because of its effects on human health and climate change. PM long-term characterisation allows identifying trends and evaluating the outcomes of environmental protection policies. This work is aimed to study the inter-annual variability of PM2.5 and PM10 concentrations and chemical composition in an urban background site (Italy). A dataset of daily PM2.5 and PM10 was collected in the period 2016–2017, including the content of OC, EC, major water-soluble ions, main metals, and compared to a similar dataset collected in the period 2013–2014. Oxidative potential using DTT assay (dithiothreitol) was evaluated and expressed in DTTV as 0.39 nmol/min·m3 in PM10 and 0.29 in PM2.5 nmol/min·m3. PM source apportionment was computed using the EPA PMF5.0 model and source contributions compared with those of a previous dataset collected between 2013 and 2014. Multi linear regression analysis identified which source contributed (p < 0.05) to the oxidative potential of each size fraction. Inter-annual trends were more evident on PM2.5 with reductions of biomass burning contribution and increases in traffic contribution in the 2016–2017 period. Crustal contributions were similar for the two periods, in both size fractions. Carbonates were comparable in PM10 with a slight increase in PM2.5. Sea spray decreased in PM10. The DTTV of PM2.5 peaked during cold periods, while, the DTTV of the PM10-2.5 fraction peaked in summer, suggesting that different sources, with different seasonality, influence OP in the PM2.5 and PM10-2.5 fractions. Analysis showed that sea spray, crustal, and carbonates sources contribute ∼13.6% to DTTV in PM2.5 and ∼62.4% to DTTV in PM10-2.5. Combustion sources (biomass burning and traffic) contribute to the majority of DTTV (50.6%) in PM2.5 and contribute for ∼26% to DTTV in PM10-2.5. Secondary nitrate contributes to DTTV in both fine and coarse fraction; secondary sulphate contribute to DTTV in PM2.5 with negligible contributions to DTTV in PM10-2.5.

Hydroxamate and thiosemicarbazone: Two highly promising scaffolds for the development of SARS-CoV-2 antivirals

The emerging COVID-19 pandemic generated by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has severely threatened human health. The main protease (Mpro) of SARS-CoV-2 is promising target for antiviral drugs, which plays a vital role for viral duplication. Development of the inhibitor against Mpro is an ideal strategy to combat COVID-19. In this work, twenty-three hydroxamates 1a-i and thiosemicarbazones 2a-n were identified by FRET screening to be the potent inhibitors of Mpro, which exhibited more than 94% (except 1c) and more than 69% inhibition, and an IC50 value in the range of 0.12–31.51 and 2.43–34.22 μM, respectively. 1a and 2b were found to be the most effective inhibitors in the hydroxamates and thiosemicarbazones, with an IC50 of 0.12 and 2.43 μM, respectively. Enzyme kinetics, jump dilution and thermal shift assays revealed that 2b is a competitive inhibitor of Mpro, while 1a is a time-dependently inhibitor; 2b reversibly but 1a irreversibly bound to the target; the binding of 2b increased but 1a decreased stability of the target, and DTT assays indicate that 1a is the promiscuous cysteine protease inhibitor. Cytotoxicity assays showed that 1a has low, but 2b has certain cytotoxicity on the mouse fibroblast cells (L929). Docking studies revealed that the benzyloxycarbonyl carbon of 1a formed thioester with Cys145, while the phenolic hydroxyl oxygen of 2b formed H-bonds with Cys145 and Asn142. This work provided two promising scaffolds for the development of Mpro inhibitors to combat COVID-19.

How to best monitor bivalirudin anticoagulant effect for ECMO and VAD-Comparison of four assay methods.

Unfractionated heparin is widely used as an anticoagulant for extracorporeal life support (ECLS) and usually monitored with activated partial thromboplastin time (aPTT). Due to its limitations in pediatric populations and interferences with monitoring, bivalirudin is being utilized more frequently in these settings. For bivalirudin, other tests have emerged such as dilute thrombin time (dTT) and ecarin chromogenic assay (ECA); however, their utilities in pediatrics are unexplored. Development of suitable, accurate testing for bivalirudin monitoring is paramount to prevent complications. We sought to compare aPTT, aPTT with heparinase (HPTT), dTT1:4, modified dTT1:10, and ECA for monitoring of pediatric ECLS patients anticoagulated with bivalirudin. aPTT, HPTT, dTT1:4, dTT1:10, and ECA were measured in 51 specimens from 17 children on bivalirudin-anticoagulated ECLS. Normal pooled plasma was spiked with various bivalirudin concentrations, and aPTT, dTT1:4, dTT1:10, and ECA were measured. In addition, dTT assays were performed using plasma from normal donors spiked with bivalirudin, heparin, and cryoprecipitate. dTT1:4 showed excellent correlation with ECA, while dTT1:4 correlated moderately with aPTT or HPTT. Fifty to 75% of specimens showed discordant results between dTT1:4 and HPTT. We found that dTT1:4 and ECA prolongations are associated with bivalirudin infusion rate; however, there are age-based differences that should be accounted for. The performance of dTT1:10 was similar to dTT1:4, though it was less sensitive to interfering factors (heparin or hyperfibrinogenemia). dTT1:10 appears to be more suitable for routine practice due to fewer variations and lower cost for monitoring bivalirudin in pediatric ECLS.

Use of Bivalirudin-Specific Monitoring Assays in Ventricular Assist Device Patients

Increasing numbers of pediatric ventricular assist device (VAD) patients are being anticoagulated with the parenteral direct thrombin inhibitor bivalirudin because it is reportedly associated with fewer bleeding and thrombotic events. With expanded use, management is shifting from a handful of experts to a wider pool of clinicians and trainees, increasing the importance of identifying broadly acceptable, standardized monitoring assays. The pharmacokinetics of bivalirudin have not been well-studied in the pediatric population and drug monitoring in all ages has been problematic for critically ill patients who require intermediate or longer-term therapeutic anticoagulation. The dilute thrombin time (dTT), available in many clinical laboratories, has been suggested as a potentially superior alternative to the activated partial thromboplastin time (aPTT), but results have been inconsistent. As clinical use of the dTT (c-dTT) for monitoring bivalirudin increased at our institution, we sought to evaluate the performance of commercially available, “research only” functional bivalirudin assays with calibrators and controls to measure bivalirudin's anticoagulation effect, utilizing residual plasmas and clinical data from VAD patients treated with bivalirudin. Residual citrated, platelet poor plasma samples from clinically ordered laboratory tests in VAD patients were collected and stored frozen at -70 oC from February 8, 2018, to January 4, 2021. With IRB approval, the samples were analyzed in conjunction with medical record review. Two experimental assay kits were utilized, ex-dTT: a dilute thrombin time assay (Hemoclot, Hyphen-Biomed, FR) and ex-anti FIIa: a chromogenic anti-factor IIa assay (BiophenDTI, Hyphen-Biomed, FR). Bivalirudin calibrators (Biophen, Hyphen-Biomed, FR) were used to develop a standard curve for the assays. Controls of low and high (1.5 and 4 microgram/mL) bivalirudin (Biophen, Hyphen-Biomed, FR) were used for quality control of the assays. Results from the two experimental assays were compared with available standard laboratory monitoring when results were available. In total, 115 residual plasma samples from 11 different patients (up to 16 samples per patient) were analyzed. Subjects included 1 adult (37 yr.) and 10 pediatric patients (0-18 yr.). There was excellent correlation between the two experimental assays (Fig. 1A). Correlation was good between the c-dTT and each of the experimental assays; however, with a clinical laboratory platform change (instrument, reagents) midway through sample collection, the c-dTT results shifted substantially in their corresponding estimate of bivalirudin concentration (Figs 1B and C). Activated partial thromboplastin time (aPTT) had poor correlation with the c-dTT and with each of the experimental assays (Fig. 1D). Here we report results from two commercially available kits that estimate bivalirudin concentration using dTT or chromogenic anti-factor IIa assays, in comparison with clinically generated results from VAD patients treated with bivalirudin. Our findings agree with previous observations that the aPTT shows poor correlation with dTT assays (clinically used and experimental) over days and weeks of anticoagulation. The two experimental assays had excellent correlation with each other and good correlation with the c-dTT; however, the fact that the c-dTT results shifted dramatically with a clinical laboratory platform change is illustrative of the need for a bivalirudin-specific monitoring assay as an essential tool for improving outcomes at our center and across centers. More research is needed to understand which type of monitoring assay (dTT or anti-FIIa) may be better suited to particular clinical circumstances. [Display omitted] DisclosuresLorts: Abbott: Consultancy; Medtronic: Consultancy; Berlin Heart: Consultancy; Syncardia: Consultancy; Abiomed: Consultancy.

Reactive oxygen species (ROS) activity of fine particulate matter health impacts in Addis Ababa, Ethiopia

Previous studies in Addis Ababa, Ethiopia, focused on PM characteristics and sources, but PM's biological activity in this area has not been adequately investigated. In this study, cellular and acellular ROS activities were measured to assess PM toxicity. PM2.5 samples in Addis Ababa were collected from November 2015 to November 2016 and their chemical composition was analyzed (organic and elemental carbon (OC/EC), ions, elements, and organic molecular markers). PM-induced ROS activity was measured in vitro in alveolar macrophage cells and using the acellular DTT assay. PM2.5 in Addis Ababa primarily consisted of primary pollutants (e.g., EC, water-insoluble OC). Annual average cellular ROS activities (±SD) were 46.5 ± 15.5 μg Zymosan/m3 and 995 ± 403 μg Zymosan/mg PM (normalized to air volume and PM2.5 mass, respectively), with no clear seasonal variations. The annual average DTT consumption rates (±SD) were 2.35 ± 0.83 nmol/min/m3 and 51.9 ± 17.6 nmol/min/mg PM, respectively, and were also similar throughout the year. Only the monthly air volume-based ROS activity had a similar trend to monthly mass concentration. Compared to other major cities worldwide, Addis Ababa PM2.5 had much lower ROS activity with no strong seasonal trends, paralleling the lack of seasonal changes in PM composition and sources. Poor correlations were observed between ROS activity and all components except mobile source tracers. This difference might be due to lower secondary aerosol content in PM2.5 in Addis Ababa than in other cities.

Comparison of indoor and outdoor oxidative potential of PM2.5: pollution levels, temporal patterns, and key constituents

Oxidative potential (OP) of PM2.5 is an emerging health indicator representing its ability to induce oxidative stress and cause adverse health effects. We examined pollution levels, temporal variations, and key constituents of PM2.5 OP by DTT assay in both indoor and outdoor environments in Nanjing, China, for over one year. Outdoor OPM (mass-normalized OP characterizes toxicity) and OPV (volume-based OP characterizes overall oxidative burden) in Nanjing were at a medium level compared to results reported for twenty-seven cities. Although PM2.5 mass concentration consistently decreased during outdoor-to-indoor transport, OPM varied by a factor of up to 2 in either direction, indicating a change of PM2.5’s ability to disrupt oxidative-reductive balance. Temporally, both outdoor and indoor OPM exhibited a significant seasonality pattern (P < 0.01) as autumn > summer > spring > winter. Outdoor and indoor daytime-nighttime OPV and OPM are fluctuating within two-fold range. In addition, the change in water-soluble Fe had the highest correlation coefficient (P < 0.05) with ΔOPM (ΔOPM = OPM, in-OPM, out) among constituents measured here. Our results suggest that development of mitigation strategies take indoor PM2.5’s OP into account, instead of outdoors only, since they differ.

Oxidative Potential, Cytotoxicity, and Intracellular Oxidative Stress Generating Capacity of PM10: A Case Study in South of Italy

Long and short-term exposure to atmospheric particulate matter (PM) has detrimental effects on human health. The effective mechanisms leading to PM toxicity are still not fully understood, even if it is known that physical-chemical properties, strongly influenced by sources and atmospheric processes, are known to play an important role. In this work, PM10 samples were collected, at an urban background site in southern Italy, to determine cytotoxicity (using MTT test on A549 cells), genotoxicity (using the comet assay), and intracellular oxidative stress on A549 cells exposed for 24 h to aqueous extracts of PM10 samples. Organic carbon (OC) and elemental carbon (EC) content of PM10 and acellular determination of oxidative potential with DTT assay were performed to compare results of acellular and cellular biological assays. Cellular (OSGCV and MTTV) and acellular (OPDTTV) outcomes, normalized in volume, are well correlated (statistically significant results) with carbon content suggesting that combustion sources play an important role in determining cellular oxidative stress and cytotoxicity of PM10. Even if the number of data is limited, genotoxicity results are well correlated (Pearson r &gt; 0.95) with OSGCV and MTTV, and a weaker, but statistically significant correlation was observed with OPDTTV. OSGCV is well correlated with the cell mortality observed with the MTTV test and a lower, but still statistically significant correlation is observed between MTTV and OPDDTV. A statistically significant correlation was found between OPDTTV and OSGCV results. When the outcomes of the cellular and acellular assay are compared normalized in mass (i.e., intrinsic values), the correlations become significantly weaker suggesting that the different sources acting on the site produces particulate matter with different toxicological potential influencing differently the biological tests studied.

Oxidative Potential Induced by Ambient Particulate Matters with Acellular Assays: A Review

Acellular assays of oxidative potential (OP) induced by ambient particulate matters (PMs) are of great significance in screening for toxicity in PMs. In this review, several typical OP measurement techniques, including the respiratory tract lining fluid assay (RTLF), ascorbate depletion assay (AA), dithiothreitol assay (DTT), chemiluminescent reductive acridinium triggering (CRAT), dichlorofluorescin assay (DCFH) and electron paramagnetic/spin resonance assay (EPR/ESR) are discussed and their sensitivity to different PMs species composition, PMs size distribution and seasonality is compared. By comparison, the DTT assay tends to be the preferred method providing a more comprehensive measurement with transition metals and quinones accumulated in the fine PMs fraction. Specific transition metals (i.e., Mn, Cu, Fe) and quinones are found to contribute OPDTT directly whereas the redox properties of PMs species may be changed by the interactions between themselves. The selection of the appropriate OP measurement methods and the accurate analysis of the relationship between the methods and PM components is conducive to epidemiological researches which are related with oxidative stress induced by PMs exposure.

Spatial mapping and size distribution of oxidative potential of particulate matter released by spatially disaggregated sources

The ability of particulate matter (PM) to induce oxidative stress is frequently estimated by acellular oxidative potential (OP) assays, such as ascorbic acid (AA) and 1,4-dithiothreitol (DTT), used as proxy of reactive oxygen species (ROS) generation in biological systems, and particle-bound ROS measurement, such as 2′,7′-dichlorodihydrofluorescein (DCFH) assay. In this study, we evaluated the spatial and size distribution of OP results obtained by three OP assays (OPAA, OPDCFH and OPDTT), to qualitative identify the relative relevance of single source contributions in building up OP values and to map the PM potential to induce oxidative stress in living organisms. To this aim, AA, DCFH and DTT assays were applied to size-segregated PM samples, collected by low-pressure cascade impactors, and to PM10 samples collected at 23 different sampling sites (about 1 km between each other) in Terni, an urban and industrial hot-spot of Central Italy, by using recently developed high spatial resolution samplers of PM, which worked in parallel during three monitoring periods (February, April and December 2017). The sampling sites were chosen for representing the main spatially disaggregated sources of PM (vehicular traffic, rail network, domestic heating, power plant for waste treatment, steel plant) present in the study area. The obtained results clearly showed a very different sensitivity of the three assays toward each local PM source. OPAA was particularly sensitive toward coarse particles released from the railway, OPDCFH was sensible to fine particles released from the steel plant and domestic biomass heating, and OPDTT was quite selectively sensitive toward the fine fraction of PM released by industrial and biomass burning sources.