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IntroductionHow to adapt the curriculum of medicine, pharmacy, and biomedical sciences to prepare future health professionals to meet the challenge of maintaining quality care in a period of socio-ecological crisis? Addressing connections between humanity and sustainable environment should include an analysis of the reciprocal influence of various ecosystems, since it is now clear that healthcare systems have an impact on ecosystems and vice versa. Here, we propose that integrating the United Nations Sustainable Development Goals (SDGs) into the curriculum could be a first step in such a transversal education.MethodsMembers of the faculty of medicine at the University of Namur, Belgium, including teaching staff of the department of medicine, pharmacy, biomedical sciences and psychology, were invited to respond anonymously to a questionnaire about their views on the feasibility of integrating the SDGs into their teaching. A subsequent survey on students’ perceptions of such teaching was conducted by student representatives.ResultsSeventy-nine percent of surveyed members of the medical faculty believe that it is possible to integrate SDGs into their lectures. However, 44–86% of them did not know how to integrate each individual goal. 94.4% of students would like SDGs to play a greater role in their education; 64.4% of them would integrate them into existing modules; 23.9% would create an optional module, and 11.9% would create a mandatory module.ConclusionSustainable Development Goals integration into the curriculum of medicine, pharmacy, and biomedical sciences is perceived as challenging in a dense teaching program. To clarify how SDGs can translate into traditional lectures, we provide for each SDG targeted applications for bachelor’s, master’s and continuing education.

BackgroundVenous thromboembolism (VTE) poses a significant global health challenge, notably exacerbated by the use of combined oral contraceptives (COCs). Evidence mainly focuses on the type of progestogen used in COCs to establish the increased risk of VTE with less data assessed on the type of estrogen used. This meta-analysis aims to assess the risk of VTE associated with COCs containing synthetic estrogens like ethinylestradiol (EE) versus natural estrogens like estradiol (E2).MethodsA systematic review and meta-analysis was conducted following the 2020 Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Literature searches were performed in December 2023 in MEDLINE and EMBASE to identify clinical studies comparing the VTE risk between COCs containing synthetic versus natural estrogens. Studies were selected through rigorous screening, and data extraction followed standardized protocols, with statistical analyses employing a random effects model.ResultsThe search yielded five relevant studies, involving over 560,000 women/time, demonstrating a significant 33% reduction in VTE risk among users of natural estrogen-based COCs compared to synthetic estrogen-based COCs (OR 0.67, 95% CI 0.51–0.87). Stratification analyses using adjusted hazard ratios (HR) of the main observationnal studies showed a 49% reduced VTE risk of E2-based pills compared to EE in association with levonorgestrel.Discussion and conclusionDespite the longstanding use of EE-based COCs, emerging evidence supports a lower thrombotic risk associated with natural estrogens. This meta-analysis substantiates the lower VTE risk associated with natural estrogen-based COCs compared to synthetic alternatives, advocating for a re-evaluation of contraceptive guidelines to prioritize patient safety and reduce thrombotic risks.

Introduction: Some studies suggest that the monovalent mRNA-1273 vaccine is more effective than BNT162b2 in producing higher levels of antibodies. However, limited data are available, and the methods used are not directly comparable. Material and methods: Blood samples were obtained before the booster (third dose) and after 14, 90, and 180 days in two similar cohorts who received the original BNT162b2 or mRNA-1273 vaccine designed to target wild type SARS-CoV-2. The aim of our study is to compare their effectiveness by assessing the levels of binding and neutralizing antibodies specifically against each of the BA.1 variant, BA.5 variant, and the XBB.1.5 subvariant. Results: Once the peak was reached after two weeks, a drastic decline in binding and neutralizing antibodies was observed up to 6 months after the homologous booster administration. The humoral response was however more sustained with the mRNA-1273 booster, with half-lives of 167, 55, and 48 days for binding, BA.1, and BA.5 neutralizing antibodies compared to 144, 30, and 29 days for the BNT162b2 booster, respectively. Compared to the BA.1 variant, the neutralizing capacity was significantly decreased at 6 months with the BA.5 variant (fold-decrease: 1.67 to 3.20) and the XBB.1.5. subvariant (fold-decrease: 2.86 to 5.48). Conclusion: Although the decrease in the humoral response was observed with both mRNA vaccines over time, a more sustained response was observed with the mRNA-1273 vaccine. Moreover, the emergence of Omicron-based variants causes a reduced neutralizing capacity, notably with the XBB.1.5. subvariant. The administration of subsequent boosters would therefore be needed to restore a sufficiently high neutralizing response.

Objectives: An increase evasion of the SARS-CoV-2 virus toward vaccination strategies and natural immunity has been rapidly described notably because of the mutations in the spike receptor binding domain and the N-terminal domain. Methods: Participants of the CRO-VAX HCP study who received the bivalent booster were followed up at 6 months. A pseudovirus‐neutralization test was used to assess the neutralization potency of antibodies against D614G, Delta, BA.1, BA.5, XBB.1.5, BA.2.86, FL.1.5.1, and JN-1. Results: The neutralizing capacity of antibodies against the Omicron variant or its subvariants was significantly reduced compared with D614G and Delta (P

AZD7442 (150 mg of tixagevimab plus 150 mg of cilgavimab) has been approved for the preexposure prophylaxis of COVID-19 and for the treatment of adults and adolescents with COVID-19 who do not require supplemental oxygen and who are at increased risk of severe COVID-19. Thus, the aim of the present study is to evaluate the neutralizing capacity of tixagevimab and cilgavimab across different SARS-CoV-2 variants in two patients who received AZD7442 for immunoprophylaxis. A cohort of subjects (n = 45) who had received the BNT162b2 mRNA COVID-19 vaccine has been included to compare these two preventive strategies. Neutralizing antibody (NAb) titers against several variants were assessed against the wild-type, alpha, beta, gamma, delta, omicron BA.5, and XBB.1.5 variants. Binding antibodies have also been measured. NAbs T1/2 for AZD7442 was 8.1 days (95% CI: 5.1–19.5 days) and was 11.8 days (95% CI: 7.9–23.7 days) for the primo-vaccination cohort. The time to reach neutralization negativity was 108.3 days (95% CI: 66.9–130.7) for AZD7442 compared to 95.4 days (95% CI: 31.0–119.7 days) for the primo-vaccination cohort. The time to reach NAbs’ negativity differs between variants with the maximum value obtained for alpha (i.e., 101.1 days (95% CI: 30.0–135.4 days)) and the minimum obtained for beta (i.e., 61.2 days (95% CI: 37.8–77.1 days)). Our results reinforce the need of reviewing the use of AZD7442 in relation to variants of concern and potentially adapting its administration schedule. AZD7442 could be indicated for short-term prophylaxis in frail patients who may be acutely exposed to SARS-CoV-2.

Safety and efficacy of vaccines against the SARS-CoV-2 coronavirus has been demonstrated in clinical trials and next by their real world use through the course of the ongoing COVID-19 pandemic. However, very rare adverse events have been detected post-authorization in certain parts of the world. This meeting report summarizes an EMA workshop’s discussion on the epidemiology, clinical presentation and biology of thrombosis with thrombocytopenia syndrome after adenovirus vector COVID-19 vaccination. General agreement was reached by international regulators, scientists and developers on the steps needed to fill the gaps in the characterization of this new syndrome. In particular, actions should be taken to improve the post-vaccination surveillance activities in low and middle income countries and investigate potential genetic predisposition factors.

Abstract Introduction The activated partial thromboplastin time (aPTT) and the prothrombin time (PT) are widely available coagulation parameters which are however poor predictors of the anticoagulant effect of direct oral anticoagulants (DOACs). Some coagulometers use the clot waveform analysis (CWA) to assess the clotting time but mainly based on a unique parameter. The improvement of these methodologies and the evaluation of the other waveform parameters may increase the sensitivity to DOACs. Objectives To assess the performance of an improved clot waveform an method (i.e. FibWave) to detect the impact of edoxaban on the coagulation and the fibrinolytic systems. Methods Seventy‐one samples from patients treated with edoxaban collected at minimum concentration (CTROUGH) and/or maximum concentration (CMAX), and 45 control samples were included. The aPTT‐ and PT‐based CWA as well as the FibIn, FibEx, and FibLysis methodologies of the FibWave were implemented and performed on an ACL‐TOP 700. Results PT and FibEx clotting time were strongly correlated to edoxaban concentration (Pearson r = 0.80 and 0.89, respectively). The FibEx clotting time allowed a better discrimination for samples with 30 and 50 ng/ml of edoxaban compared to PT (cutoffs of 96.5 and 114.2 s for the FibEx versus a unique cutoff of 13.1 s for the PT). The fibrinolytic process was impaired in the presence of edoxaban in a dose‐dependent manner. Conclusion FibEx is more sensitive than aPTT‐ and PT‐based CWA for the detection of the clinically relevant anticoagulant level of edoxaban.

The rapid and large-scale roll-out of new COVID-19 vaccines has led to unprecedented challenges in assessing vaccine safety. In 2021, the European Medicines Agency (EMA) processed about 1.7 million safety reports related to COVID-19 vaccines in the EudraVigilance (EV) database and identified more than 900 potential signals. Beyond the large amount of information to be processed, the evaluation of safety signals has faced several difficulties and limitations, both in the assessment of case reports and in the investigation of databases. The evaluation of a signal of corneal graft rejection (CGR) with Vaxzevria® was no exception to this. In this commentary, we present the challenges encountered in making regulatory decisions in the context of evolving evidence and knowledge. The pandemic crisis emphasised the importance of quick and proactive communication to address the many questions and, above all, to ensure the transparency of safety data.

Several studies reported on the humoral response in subjects having received the BNT162b2 mRNA COVID-19 vaccine. However, data on the kinetics of antibodies 3 months post-vaccination are currently lacking and are important to drive the future vaccination strategy. The CRO-VAX HCP study is an ongoing multicentre, prospective and interventional study designed to assess the antibody response in a population of healthcare professionals who had received two doses of the BNT162b2 mRNA COVID-19 vaccine. Two hundred individuals underwent a blood drawn within 2 days before the first vaccine dose. One-hundred and forty-two persons (71%) were categorized as seronegative at baseline while 58 (29%) were seropositive. Samples were then collected after 14, 28, 42, 56, and 90 days. Antibodies against the SARS-CoV-2 nucleocapsid and the receptor binding domain of the S1 subunit of the spike protein were measured in all individuals at different time points. Using a one-compartment kinetics model, the time to maximum concentration was estimated at 36 ± 3 days after the first dose and the estimated half-life of antibodies was 55 days (95% CI: 37–107 days) in seronegative participants. In seropositive participants, the time to maximum concentration was estimated at 24 ± 4 days and the estimated half-life was 80 days (95% CI: 46–303 days). The antibody response was higher in seropositive compared to seronegative participants. In both seropositive and seronegative subjects, a significant antibody decline was observed at 3 months compared to the peak response. Nevertheless, the humoral response remained robust in all participants.

Abstract Introduction Edoxaban is the only anti‐Xa inhibitor metabolized in pharmacologically active moiety that could interfere with chromogenic anti‐Xa assays, especially in case of drug‐drug interactions or physiological disorders. Materials and methods We evaluated the contribution of the main metabolite of edoxaban, edoxaban‐M4 (M4), in 79 plasma samples from patients taking edoxaban. The total anti‐Xa activity was evaluated on three different chromogenic factor Xa‐based assays. Results were compared with a validated ultra‐high‐performance liquid chromatography coupled with a tandem mass spectrometry measurement. Edoxaban and its active M4 metabolite have also been spiked separately in normal pooled plasma to assess the sensitivity of chromogenic anti‐Xa assays to both molecules individually. Results Spiked edoxaban or M4 provided different slopes of linear regression models between chromogenic and chromatographic measurement (from 0.97 for STA Liquid Anti‐Xa to 1.10 for Biophen Heparin LRT Low with edoxaban and from 0.70 for Biophen DiXaI High to 0.83 for Biophen Heparin LRT High, respectively). A positive correlation is observed between the increase of the ratio M4/edoxaban with the difference between chromogenic and chromatographic measurements. Conclusion Edoxaban and M4 do not similarly impact chromogenic assays, leading to biased chromogenic estimations of ponderal concentrations. In patient samples, this impact is even more important at low concentrations or in the case of an increase in the M4/edoxaban ratio because of hepatic or renal impairments or in case of drug interactions. This study highlights the limitations and risks of error of expressing results in ponderal concentrations instead of global activity anti‐Xa.

Abstract Background The evaluation of activated protein C (APC) resistance based on the endogenous thrombin potential (ETP) is recommended during the development of steroid contraceptives in women. In 2019, this assay was validated on the calibrated automated thrombogram (CAT) device. However, in view of its screening potential, its automation is essential. Objectives To transfer the ETP‐based APC resistance assay on the ST Genesia system using reagent STG‐ThromboScreen with exogenous APC added. Method Dose‐response curves were performed to define APC concentration leading to 90% ETP inhibition on healthy donors. Intra‐ and interrun reproducibility was assessed. The normal range was defined on the basis of 56 samples from healthy individuals. The sensitivity was assessed on 40 samples from women using combined oral contraceptives (COCs). A method comparison with the validated ETP‐based APC resistance on the CAT system was performed. Results were expressed in normalized APC sensitivity ratio (nAPCsr). Results The APC concentration leading to 90% ETP inhibition was 652 mU/mL. Intra‐ and interrun reproducibility showed standard deviation

Many factors must be considered and discussed with women when initiating a contraceptive method and the risk of venous thromboembolism (VTE) is one of them. In this review, we discuss the numerous strategies that have been implemented to reduce the thrombotic risk associated with combined oral contraceptives (COCs) from their arrival on the market until today. Evidences suggesting that COCs were associated with an increased risk of VTE appeared rapidly after their marketing. Identified as the main contributor of this risk, the dosage of the estrogen, i.e., ethinylestradiol (EE), was significantly reduced. New progestins were also synthetized (e.g., desogestrel or gestodene) but their weak androgenic activity did not permit to counterbalance the effect of EE as did the initial progestins such as levonorgestrel. Numerous studies assessed the impact of estroprogestative combinations on hemostasis and demonstrated that women under COC suffered from resistance towards activated protein C (APC). Subsequently, the European Medicines Agency updated its guidelines on clinical investigation of steroid contraceptives in which they recommended to assess this biological marker. In 2009, estradiol-containing COCs were marketed and the use of this natural form of estrogen was found to exert a weaker effect on the synthesis of hepatic proteins compared to EE. In this year 2021, a novel COC based on a native estrogen, i.e., estetrol, will be introduced on the market. Associated with drospirenone, this preparation demonstrated minor effects on coagulation proteins as compared with other drospirenone-containing COCs. At the present time, the standard of care when starting a contraception, consists of identifying the presence of hereditary thrombophilia solely on the basis of familial history of VTE. This strategy has however been reported as poorly predictive of hereditary thrombophilia. One rationale and affordable perspective which has already been considered in the past could be the implementation of a baseline screening of the prothrombotic state to provide health care professionals with objective data to support the prescription of the more appropriate contraceptive method. While this strategy was judged too expensive due to limited laboratory solutions, the endogenous thrombin potential-based APC resistance assay could now represent an interesting alternative.

Abstract Background and objective Although the endogenous thrombin potential (ETP)‐based activated protein C (APC) resistance is recommended for the development of steroid contraceptive agents, one of the main limitations of this technique was its lack of standardization, which hampered study‐to‐study comparison. A validated methodology that meets all the regulatory requirements in terms of analytical performances has been developed recently. To ensure a wide implementation of this test, the assessment of the interlaboratory variability was needed. Method The assay was implemented in three testing laboratories. First, dose‐response curves were performed to locally define APC concentration leading to 90% of ETP inhibition on healthy donors. Intra‐ and inter‐run repeatability were assessed on a reference plasma and three quality controls. To investigate the variability in results among the different testing units, 60 donor samples were analyzed at each site. Results The APC concentration leading to 90% of ETP inhibition was defined at 1.21 µg/ml and 1.14 µg/ml in the two receiving units. Intra‐ and inter‐run repeatability showed standard deviation below 3%. Analyses of the 60 donor samples showed no statistically significant difference. The sensitivity of the test in the different laboratories was maintained and subgroup analyses still reported significant differences depending on hormonal status of donors. Conclusion This study is the first reporting the interlaboratory variability of the ETP‐based APC resistance assay. Data revealed excellent intra‐ and interlaboratory reproducibility. These results support the concept that this blood coagulation test provides an appropriate sensitivity irrespective of the laboratory in which analyses are performed.

Background: in this report, we describe the case of an 83-year-old woman vaccinated with ChadOx1 nCoV-19 who developed a so-called vaccine-induced thrombosis with thrombocytopenia syndrome and who did not develop any antibodies against the spike protein of SARS-CoV-2 at 30 days following the administration of her first dose of ChadOx1 nCoV-19. Experimental section: two serum samples from the patient and 5 serum samples from 5 control individuals having received the two-dose regimen vaccination with ChadOx1 nCoV-19 were evaluated. In order to investigate the lack of response to the vaccination, a cell model was developed. This model permits to evaluate the interaction between responsive cells (A549) possessing the Coxsackievirus and Adenovirus Receptor (CAR), a defined concentration of ChadOx1 nCoV-19 and serial dilution of the patient or the control serum. The aim was to assess the impact of these sera on the production of the spike (S) protein induced by the transfection of the genetic material of ChadOx1 nCoV-19 into the A549 cells. The S protein is measured in the supernatant using an ELISA technique. Results: interestingly, the serum from the patient who developed the vaccine-induced thrombosis with thrombocytopenia syndrome impaired the production of S protein by the A549 cells transfected with ChadOx1 nCoV-19. This was not observed with the controls who did not interfere with the transfection of ChadOx1 nCoV-19 into A549 cells since the S protein is retrieved in the supernatant fraction. Conclusion: based on the data coming from the clinical and the cell model information, we found a possible explanation on the absence of antibody response in our patient. She has, or has developed, characteristics that prevent the production of the S protein in contrast to control subjects. We were not able to investigate the entire mechanism behind this resistance which deserve further investigations. A link between this resistance and the development of the thrombosis with thrombocytopenia syndrome following vaccination with ChadOx1 nCoV-19 cannot be excluded.

NETosis is a form of neutrophil death leading to the release of extracellular chromatin and the assembling of proteins, including antiviral proteins, primed by an initial pathogenic stimulus. Under certain specific conditions, neutrophils can exhibit a double-edged activity. This event has been implicated in COVID-19 among other conditions. Neutrophil extracellular traps (NETs) are involved in the pathogenesis of COVID-19 by promoting a pro-inflammatory and a procoagulant state leading to multiorgan failure. This particular form of host defense promoted by neutrophils is closely related to the well-known cytokine storm observed in severe COVID-19 patients. These two elements therefore represent possible targets for treatment of severe SARS-CoV-2 infections.

The diagnostic of SARS-CoV-2 infection relies on reverse transcriptase polymerase chain reactions (RT-PCRs) performed on nasopharyngeal (NP) swabs. Nevertheless, false-negative results can be obtained with inadequate sampling procedures, making the use of other biological matrices worthy of investigation. This study aims to evaluate the kinetics of serum N antigens in severe and non-severe patients and compare the clinical performance of serum antigenic assays with NP RT-PCR. Ninety patients were included in the study and monitored for several days. Disease severity was determined according to the WHO clinical progression scale. Serum N antigen levels were measured with a chemiluminescent assay (CLIA) and the Single Molecular Array (Simoa) assay. Viremia thresholds for severity were determined and proposed. In severe patients, the peak antigen response was observed 7 days after the onset of symptoms, followed by a decline. No real peak response was observed in non-severe patients. Severity thresholds for the Simoa and the CLIA provided positive likelihood ratios of 30.0 and 10.9 for the timeframe between day 2 and day 14, respectively. Sensitive detection of N antigens in serum may thus provide a valuable new marker for COVID-19 diagnosis and evaluation of disease severity. When assessed during the first 2 weeks since the onset of symptoms, it may help in identifying patients at risk of developing severe COVID-19 to optimize better intensive care utilization.

This data article accompanies the manuscript entitled: “Prothrombotic Disturbances of hemostasis of Patients with Severe COVID-19: a Prospective Longitudinal Observational Cohort Study” submitted to Thrombosis Research by the same authors. We report temporal changes of plasma levels of an extended set of laboratory parameters during the ICU stay of the 21 COVID-19 patients included in the monocentre cohort: CRP, platelet count, prothrombin time; Clauss fibrinogen and clotting factors II, V and VIII levels, D-dimers, antithrombin activity, protein C, free protein S, total and free tissue factor pathway inhibitor, PAI-1 levels, von Willebrand factor antigen and activity, ADAMTS-13 (plasma levels); and of two integrative tests of coagulation (thrombin generation with ST Genesia) and fibrinolysis (global fibrinolytic capacity - GFC). Regarding hemostasis, we used double-centrifuged frozen citrated plasma prospectively collected after daily performance of usual coagulation tests. Demographic and clinical characteristics of patients and thrombotic and hemorrhagic complications were also collected from patient's electronic medical reports.

Rapid antigen detection (RAD) tests are commonly used for the diagnosis of SARS-CoV-2 infections. However, with the continuous emergence of new variants of concern (VOC), presenting various mutations potentially affecting the nucleocapsid protein, the analytical performances of these assays should be frequently reevaluated. One hundred and twenty samples were selected and tested with both RT-qPCR and six commercial RAD tests that are commonly sold in Belgian pharmacies. Of these, direct whole-genome sequencing identified the strains present in 116 samples, of which 70 were Delta and 46 were Omicron (BA.1 and BA.1.1 sub-lineages, respectively). The sensitivity across a wide range of Ct values (13.5 to 35.7; median = 21.3) ranged from 70.0% to 92.9% for Delta strains and from 69.6% to 78.3% for Omicron strains. When taking swabs with a low viral load (Ct > 25, corresponding to 10 copies/mL), only the Roche RAD test showed acceptable performances for the Delta strains (80.0%), while poor performances were observed for the other RAD tests (20.0% to 40.0%). All the tested devices had poor performances for the Omicron samples with a low viral load (0.0% to 23.1%). The poor performances observed with low viral loads, particularly for the Omicron strain, is an important limitation of RAD tests, which is not sufficiently highlighted in the instructions for use of these devices.

BCR-ABL tyrosine kinase inhibitors (TKIs) revolutionized the treatment of chronic myeloid leukemia, inducing deep molecular responses, largely improving patient survival and rendering treatment-free remission possible. However, three of the five BCR-ABL TKIs, dasatinib, nilotinib, and ponatinib, increase the risk of developing arterial thrombosis. Prior investigations reported that nilotinib and ponatinib affect the endothelium, but the mechanisms by which they exert their toxic effects are still unclear. The impact of dasatinib and bosutinib on endothelial cells has been poorly investigated. Here, we aimed to provide an in vitro homogenous evaluation of the effects of BCR-ABL TKIs on the endothelium, with a special focus on the type of cell death to elucidate the mechanisms responsible for the potential cytotoxic effects of BCR-ABL TKIs nilotinib and ponatinib on endothelial cells. We tested the five BCR-ABL TKIs at three concentrations on human umbilical venous endothelial cells (HUVECs). This study highlights the endothelial toxicity of ponatinib and provides insights about the mechanisms by which it affects endothelial cell viability. Ponatinib induced apoptosis and necrosis of HUVECs after 72 h. Dasatinib affected endothelial cells in vitro by inhibiting their proliferation and decreased wound closure as soon as 24 h of treatment and even at infra-therapeutic dose (0.005 µM). Comparatively, imatinib, nilotinib, and bosutinib had little impact on endothelial cells at therapeutic concentrations. They did not induce apoptosis nor necrosis, even after 72 h of treatment but they inhibited HUVEC proliferation. Overall, this study reports various effects of BCR-ABL TKIs on endothelial cells and suggests that ponatinib and dasatinib induce arterial thrombosis through endothelial dysfunction.

Abstract Background Serious bleeding events have been frequently described in patients taking direct oral anticoagulants (DOAC). In secondary analyses of phase 3 trials, DOAC plasma concentrations were shown to correlate with bleeding outcomes. This study aimed to describe rivaroxaban plasma levels in patients admitted to the emergency department (ED) for bleeding events. For each patient, risk factors for experiencing bleeding events were also investigated. Methods This analysis was part of an observational study conducted in the ED of two teaching hospitals. Plasma samples from 10 rivaroxaban-treated patients admitted for bleeding events were collected. Rivaroxaban plasma concentrations were determined by calibrated chromogenic anti-Xa assay. The measured rivaroxaban levels were then extrapolated at trough using a published population pharmacokinetic (PopPK) model, and compared to on-therapy ranges observed in large clinical trials. For each patient, clinical, medication and ABCB1 genotype data were collected. Results Rivaroxaban measurements varied from 5 to 358 ng/ml, with a post-intake delay ranging from 9 to 38 h. At trough, estimated plasma concentrations were between 12 and 251 ng/ml (median value 94 ng/ml). Four patients had higher-than-expected rivaroxaban levels. Inadequate dose regimen, excessive alcohol consumption and lack of treatment reassessment were observed in several patients. Half of patients were taking ≥1 drug with potential pharmacokinetics interactions (e.g. amiodarone, diltiazem), while half of patients were taking ≥1 drug increasing the risk of bleeding. All 3 patients with available genotyping data and higher-than-expected rivaroxaban levels were heterozygous or homozygous mutated for the ABCB1 1236C > T, 2677G > T, 3435 C > T and rs4148738 single nucleotide polymorphisms (SNP). Conclusions Rivaroxaban patients admitted to the ED for bleeding events showed highly variable plasma concentrations. This analysis underlines the usefulness of rapid DOAC measurement and the value of PopPK models to estimate concentrations at trough in a context where the post-intake delay is unmanageable. Close patient follow-up, including renal function assessment and drug interactions review, is essential for bleeding risk minimization.

Abstract Inorganic transition metal dichalcogenide nanostructures are interesting for several biomedical applications such as coating for medical devices (e.g. endodontic files, catheter stents) and reinforcement of scaffolds for tissue engineering. However, their impact on human blood is unknown. A unique nanomaterial surface-engineering chemical methodology was used to fabricate functional polyacidic polyCOOH inorganic nanotubes of tungsten disulfide towards covalent binding of any desired molecule/organic species via chemical activation/reactivity of this former polyCOOH shell. The impact of these nanotubes on hemolysis, platelet aggregation and blood coagulation has been assessed using spectrophotometric measurement, light transmission aggregometry and thrombin generation assays. The functionalized nanotubes do not induce hemolysis but decrease platelet aggregation and induce coagulation through intrinsic pathway activation. The functional nanotubes were found to be more thrombogenic than the non-functional ones, suggesting lower hemocompatibility and increased thrombotic risk with functionalized tungsten disulfide nanotubes. These functionalized nanotubes should be used with caution in blood-contacting devices.

Neutralising antibodies (NAbs) represent the real source of protection against SARS-CoV-2 infections by preventing the virus from entering target cells. The gold standard in the detection of these antibodies is the plaque reduction neutralization test (PRNT). As these experiments must be done in a very secure environment, other techniques based on pseudoviruses: pseudovirus neutralization test (pVNT) or surrogate virus neutralization test (sVNT) have been developed. Binding assays, on the other hand, measure total antibodies or IgG, IgM, and IgA directed against one epitope of the SARS-CoV-2, independently of their neutralizing capacity. The aim of this study is to compare the performance of six commercial binding assays to the pVNT and sVNT. In this study, we used blood samples from a cohort of 62 RT-PCR confirmed COVID-19 patients. Based on the results of the neutralizing assays, adapted cut-offs for the binding assays were calculated. The use of these adapted cut-offs does not permit to improve the accuracy of the serological assays and we did not find an adapted cut-off able to improve the capacity of these tests to detect NAbs. For a part of the population, a longitudinal follow-up with at least two samples for the same patient was performed. From day 14 to day 291, more than 75% of the samples were positive for NAbs (n = 87/110, 79.1%). Interestingly, 6 months post symptoms onset, the majority of the samples (N = 44/52, 84.6%) were still positive for NAbs. This is in sharp contrast with the results we obtained 6 months post-vaccination in our cohort of healthcare workers who have received the two-dose regimens of BNT162b2. In this cohort of vaccinated subjects, 43% (n = 25/58) of the participants no longer exhibit NAbs activity 180 days after the administration of the first dose of BNT162b2.

Data about the long-term duration of antibodies after SARS-CoV-2 vaccination are still scarce and are important to design vaccination strategies. In this study, 231 healthcare professionals received the two-dose regimen of BNT162b2. Of these, 158 were seronegative and 73 were seropositive at baseline. Samples were collected at several time points. The neutralizing antibodies (NAbs) and antibodies against the nucleocapsid and the spike protein of SARS-CoV-2 were measured. At day 180, a significant antibody decline was observed in seronegative (−55.4% with total antibody assay; −89.6% with IgG assay) and seropositive individuals (−74.8% with total antibody assay; −79.4% with IgG assay). The estimated half-life of IgG from the peak humoral response was 21 days (95% CI: 13–65) in seronegative and 53 days (95% CI: 40–79) in seropositive individuals. The estimated half-life of total antibodies was longer and ranged from 68 days (95% CI: 54–90) to 114 days (95% CI: 87–167) in seropositive and seronegative individuals, respectively. The decline of NAbs was more pronounced (−98.6%) and around 45% of the subjects tested were negative at day 180. Whether this decrease correlates with an equivalent drop in the clinical effectiveness against the virus would require appropriate clinical studies.

This case reports on the successful maternal to fetal transfer of neutralizing antibodies after vaccination with BNT162b2 in a pregnant woman at 25 weeks of gestation. The levels of neutralizing antibodies were approximately 5-fold higher in the umbilical cord than in the maternal blood while the level of total antibodies showed only a 2-fold increase. This suggest that the antibodies that crossed the syncytiotrophoblast cell barrier have specific characteristics that correlate to functional neutralizing capacity. Although pregnant and lactating women have been excluded from clinical trials for several reasons including ethical concerns about fetal exposure, accumulating evidence has now revealed that these vaccines are safe and efficient for both the fetus and the woman. Vaccination against COVID-19 in pregnancy is vital to control disease burden and to decrease morbidity in the ante-, peri- and post-natal periods. Inclusion of pregnant women in research programs for the development of SARS-CoV-2 vaccines should be mandatory to provide this population with the equitable benefits of vaccine research.

The evaluation of the neutralizing capacity of anti-SARS-CoV-2 antibodies is important because they represent real protective immunity. In this study we aimed to measure and compare the neutralizing antibodies (NAbs) in COVID-19 patients and in vaccinated individuals. One-hundred and fifty long-term samples from 75 COVID-19 patients were analyzed with a surrogate virus neutralization test (sVNT) and compared to six different SARS-CoV-2 serology assays. The agreement between the sVNT and pseudovirus VNT (pVNT) results was found to be excellent (i.e., 97.2%). The NAb response was also assessed in 90 individuals who had received the complete dose regimen of BNT162b2. In COVID-19 patients, a stronger response was observed in moderate–severe versus mild patients (p-value = 0.0006). A slow decay in NAbs was noted in samples for up to 300 days after diagnosis, especially in moderate–severe patients (r = −0.35, p-value = 0.03). In the vaccinated population, 83.3% of COVID-19-naive individuals had positive NAbs 14 days after the first dose and all were positive 7 days after the second dose, i.e., at day 28. In previously infected individuals, all were already positive for NAbs at day 14. At each time point, a stronger response was observed for previously infected individuals (p-value < 0.05). The NAb response remained stable for up to 56 days in all participants. Vaccinated participants had significantly higher NAb titers compared to COVID patients. In previously infected vaccine recipients, one dose might be sufficient to generate sufficient neutralizing antibodies.

Background: Little is known about potential confounding factors influencing the humoral response in individuals having received the BNT162b2 vaccine. Methods: Blood samples from 231 subjects were collected before and 14, 28, and 42 days following coronavirus disease 2019 (COVID-19) vaccination with BNT162b2. Anti-spike receptor-binding-domain protein (anti-Spike/RBD) immunoglobulin G (IgG) antibodies were measured at each time-point. Impact of age, sex, childbearing age status, hormonal therapy, blood group, body mass index and past-history of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection were assessed by multivariable analyses. Results and Conclusions: In naïve subjects, the level of anti-Spike/RBD antibodies gradually increased following administration of the first dose to reach the maximal response at day 28 and then plateauing at day 42. In vaccinated subjects with previous SARS-CoV-2 infection, the plateau was reached sooner (i.e., at day 14). In the naïve population, age had a significant negative impact on anti-Spike/RBD titers at days 14 and 28 while lower levels were observed for males at day 42, when corrected for other confounding factors. Body mass index (BMI) as well as B and AB blood groups had a significant impact in various subgroups on the early response at day 14 but no longer after. No significant confounding factors were highlighted in the previously infected group.

Abstract Direct oral anticoagulants (DOACs) have been licensed worldwide for several years for various indications. Each year, 10–15% of patients on oral anticoagulants will undergo an invasive procedure and expert groups have issued several guidelines on perioperative management in such situations. The perioperative guidelines have undergone numerous updates as clinical experience of emergency management has increased and perioperative studies including measurement of residual anticoagulant levels have been published. The high inter-patient variability of DOAC plasma levels has challenged the traditional recommendation that perioperative DOAC interruption should be based only on the elimination half-life of DOACs, especially before invasive procedures carrying a high risk of bleeding. Furthermore, recent publications have highlighted the potential danger of heparin bridging use when DOACs are stopped before an invasive procedure. As antidotes are progressively becoming available to manage severe bleeding or urgent procedures in patients on DOACs, accurate laboratory tests have become the standard to guide their administration and their actions need to be well understood by clinicians. This review aims to provide a systematic approach to managing patients on DOACs, based on recent updates of various perioperative guidance, and highlighting the advantages and limits of recommendations based on pharmacokinetic properties and laboratory tests.

Several studies have described the long-term kinetics of anti-SARS-CoV-2 antibodies but long-term follow-up data, i.e., >6 months, are still sparse. Additionally, the literature is inconsistent regarding the waning effect of the serological response. The aim of this study was to explore the temporal dynamic changes of the immune response after SARS-CoV-2 infection in hospitalized and non-hospitalized symptomatic patients over a period of 10 months. Six different analytical kits for SARS-CoV-2 antibody detection were used. Positivity rates, inter-assay agreement and kinetic models were determined. A high inter-individual and an inter-methodology variability was observed. Assays targeting total antibodies presented higher positivity rates and reached the highest positivity rates sooner compared with assays directed against IgG. The inter-assay agreement was also higher between these assays. The stratification by disease severity showed a much-elevated serological response in hospitalized versus non-hospitalized patients in all assays. In this 10-month follow-up study, serological assays showed a clinically significant difference to detect past SARS-CoV-2 infection with total antibody assays presenting the highest positivity rates. The waning effect reported in several studies should be interpreted with caution because it could depend on the assay considered.

Direct oral anticoagulants (DOAC) are used in several indications for the prevention and treatment of thrombotic events. As highlighted by data from clinical trials and case studies, all DOAC carry the risk of bleeding despite careful selection and patient management. Previous publications have demonstrated the limited knowledge of many physicians concerning the indications for, and correct management of, these anticoagulants. Health institutions should develop risk minimisation strategies and educational materials to prevent major adverse events related to DOAC administration. Major bleeding events are reported in clinical practice and specific antidotes are emerging from Phase III trials. Some antidotes are licensed but their high cost might limit routine use. We therefore illustrate approaches and tools that can help physicians prescribe DOAC appropriately. We focus on screening for modifiable bleeding risk factors and adapting doses according to the individual benefit-risk profile. We also provide recommendations on managing a missed dose, switching, bridging, and resumption.

Standardization, data mining techniques, and comparison to normality are changing the landscape of multiparameter flow cytometry in clinical hematology. On the basis of these principles, a strategy was developed for measurable residual disease (MRD) assessment. Herein, suspicious cell clusters are first identified at diagnosis using a clustering algorithm. Subsequently, automated multidimensional spaces, named “Clouds”, are created around these clusters on the basis of density calculations. This step identifies the immunophenotypic pattern of the suspicious cell clusters. Thereafter, using reference samples, the “Abnormality Ratio” (AR) of each Cloud is calculated, and major malignant Clouds are retained, known as “Leukemic Clouds” (L-Clouds). In follow-up samples, MRD is identified when more cells fall into a patient’s L-Cloud compared to reference samples (AR concept). This workflow was applied on simulated data and real-life leukemia flow cytometry data. On simulated data, strong patient-dependent positive correlation (R2 = 1) was observed between the AR and spiked-in leukemia cells. On real patient data, AR kinetics was in line with the clinical evolution for five out of six patients. In conclusion, we present a convenient flow cytometry data analysis approach for the follow-up of hematological malignancies. Further evaluation and validation on more patient samples and different flow cytometry panels is required before implementation in clinical practice.

Imatinib, the first-in-class BCR-ABL tyrosine kinase inhibitor (TKI), had been a revolution for the treatment of chronic myeloid leukemia (CML) and had greatly enhanced patient survival. Second- (dasatinib, nilotinib, and bosutinib) and third-generation (ponatinib) TKIs have been developed to be effective against BCR-ABL mutations making imatinib less effective. However, these treatments have been associated with arterial occlusive events. This review gathers clinical data and experiments about the pathophysiology of these arterial occlusive events with BCR-ABL TKIs. Imatinib is associated with very low rates of thrombosis, suggesting a potentially protecting cardiovascular effect of this treatment in patients with BCR-ABL CML. This protective effect might be mediated by decreased platelet secretion and activation, decreased leukocyte recruitment, and anti-inflammatory or antifibrotic effects. Clinical data have guided mechanistic studies toward alteration of platelet functions and atherosclerosis development, which might be secondary to metabolism impairment. Dasatinib, nilotinib, and ponatinib affect endothelial cells and might induce atherogenesis through increased vascular permeability. Nilotinib also impairs platelet functions and induces hyperglycemia and dyslipidemia that might contribute to atherosclerosis development. Description of the pathophysiology of arterial thrombotic events is necessary to implement risk minimization strategies.

Extracellular vesicles (EVs) generated during tumourigenesis are thought to play a major role in the hypercoagulant state observed in cancer patients. They exhibit negatively charged phospholipids and tissue factor (TF) that promote coagulation cascade activation. In addition, they contain surface proteins and cytoplasmic molecules, both originating from the producing cell that can impact target cells’ expression. By targeting endothelial cells of blood vessels, these EVs could disturb the physiological anticoagulant properties of these cells and be partly responsible for the vascular endothelium activation observed in cancer patients. Indeed, vascular endothelium naturally exhibits heparin-like proteoglycan, TF pathway inhibitor and protein C anticoagulant pathway that prevent thrombosis in physiological condition. An overexpression of TF and a decreased expression of coagulation cascade inhibitors have been reported after EVs’ treatment of endothelial cells. The induction of apoptosis and an increased expression of platelet adhesion molecules have also been highlighted. These events may promote thrombus formation in cancer. The aim of this paper is to provide a targeted review on the current evidence and knowledge of roles and impact of EVs on endothelial surface anticoagulant and procoagulant factors and cellular adhesion molecules expression.

Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired disorder of the haematopoietic stem cell that makes blood cells more sensitive to the action of complement. PNH patients experience an increased risk of arterial and venous thrombosis – major causes of death due to this disease. Though many potential interlaced mechanisms are suspected, extracellular vesicles (EVs) of various origins may play a central role. The processes possibly involved are haemolysis, platelet activation, injured endothelial cells and monocyte activation. The impact of transfusion should be evaluated. A better understanding of the mechanisms involved may help to propose guidelines for the prophylaxis and treatment of thrombosis in PNH. In this paper, we propose an updated review of the pathophysiology of the underlying mechanisms of thrombosis associated with PNH, with specific focus on the prominent role of EVs.

Background: Titanium carbide (TiC) is used for ceramic metal composites in several industries and is regarded as a nanomaterial for catalyst and battery applications. However, there are very few studies in regard to the toxicological potential of TiC nanoparticles (NPs). Objective: To study the toxicodynamics and toxicokinetics of TiC NPs in Sprague Dawley rats in acute (24 h) and subacute (28 days) oral administrations. The acute doses were 0.5, 5, 50, 300 and 1000 mg kg−1; the subacute doses were 0.5 and 50 mg kg−1. Results: Organ histopathological examination (esophagus, stomach, intestines, spleen, liver, and kidneys) indicates the absence of damage at all applied doses, in both assessments. In the acute administration, alkaline phosphatases increased (5, 300 and 1000 mg kg−1), ASAT increased (1000 mg kg−1) and bile salts decreased (0.5 mg kg−1). No alterations in urine parameters (sodium, potassium, osmolarity) were found. Acute administration of TiC caused mineral changes in organs (liver, spleen, kidneys). TiC was mostly cleared by feces excretion 24 h after administration, in subacute administration causing variations in mineral absorption (Mg, Al, P, S, Ca, Zn). TiC could pass the intestinal barrier as TiC traces were detected in urine. Conclusion: No sign of toxicity was found after oral administration. TiC was excreted mostly in feces producing mineral absorption alterations. Low traces were retrieved in urine, indicating that TiC can cross the intestinal barrier.

Introduction: Extracellular vesicles (EVs) are shed from cells and carry markers of the parent cells. Vesicles derived from cancer cells reach the bloodstream and locally influence important physiological processes. It has been previously shown that procoagulant vesicles are circulating in patients’ fluids. These EVs are therefore considered as promising biomarkers for the thrombotic risk. Because of their small size, classical methods such as flow cytometry suffer from limitation for their characterisation. Atomic force microscopy (AFM) has been proposed as a promising complementary method for the characterisation of EVs. Objectives: The objectives of this study are: (a) to develop and validate AFM with specific antibodies (anti-TF) and (b) to compare air and liquid modes for EVs’ size and number determination as potential biomarkers of the prothrombotic risk. Methods: AFM multimode nanoscope III was used for air tapping mode (TM). AFM catalyst was used for liquid Peak Force Tapping (PFT) mode. Vesicles are generated according to Davila et al.'s protocol. Substrates are coated with various concentrations of antibodies, thanks to ethanolamine and glutaraldehyde. Results: Vesicles were immobilised on antibody-coated surfaces to select tissue factor (TF)-positive vesicles. The size range of vesicles observed in liquid PFT mode is 6–10 times higher than in air mode. This corresponds to the data found in the literature. Conclusion: We recommend liquid PFT mode to analyse vesicles on 5 µg/ml antibody-coated substrates.

Introduction. Patients with cancer have a 7- to 10-fold increased risk of developing venous thromboembolism. Circulating microvesicles could be a useful predictive biomarker for venous thromboembolism in cancer. Validated and standardised techniques that could be used to determine the complete microvesicle phenotype are required. These were two-fold: a) to characterise tissue factor (TF)-bearing microvesicles released by cultured breast cancer cells MDA-MB-231 by flow cytometry (FCM), transmission electron microscopy (TEM) and thrombin generation assay (TGA); and b) to validate the sensitivity and variability intra/inter-assay of TGA as a useful method to study the procoagulant activity (PCA) of microvesicles. Methods. Cultured breast cancer cells MDA-MB-231 were incubated for 45 minutes at 37°C. Samples were then centrifuged or not at 4,500 g for 15 minutes, and cells and MVs or MV-containing supernatants were used for TEM, FCM and TGA. In activity assays, microvesicles (i.e. cell-depleted supernatants) were incubated with anti-TF antibodies or with annexin V to assess the contribution of TF and phospholipids to the PCA. Alternatively, supernatants were filtered through 0.1, 0.22, 0.45 or 0.65 µm membranes and subjected to TGA. Results. The majority of the PCA was associated with microvesicles smaller than 0.1 µm, and the mean microvesicle size estimated by TEM after 10,000 g centrifugation was 121±54 nm with a majority of vesicles between 100 and 200 nm. Microvesicles derived from 5,000 MDA-MB-231cells/ml were sufficient to significantly increase the thrombin generation of normal pooled plasma. Conclusions. TEM, FCM and filtration coupled to TGA represent a useful combination to study the PCA of TF-bearing microvesicles, whatever their size. And it will be interesting to implement these techniques in patients.

Endothelial dysfunction is considered to be an early event in atherosclerosis and plays a pivotal role in the development, progression and clinical complications of atherosclerosis. Previous studies have shown the beneficial effects of combined inhibition of thromboxane synthase and antagonism of thromboxane receptors by BM-573 on atherosclerosis; however our knowledge about the beneficial effects of BM-573 on endothelial function and increased blood pressure related to early stage of atherosclerosis is limited. In the present study, we investigated the effects of short-term (3 μM, 1 hour) and chronic (10 mg/L, 8 weeks) treatments with BM-573 on vasodilatory function, nitric oxide (NO) bioavailability, oxidative stress and systolic blood pressure in 15 weeks old apolipoprotein E-deficient (ApoE-KO) mice. ApoE-KO mice showed a reduced endothelium-derived relaxation. In addition, NO bioavailability was reduced and oxidative stress and blood pressure were increased in ApoE-KO mice versus wild-type mice. BM-573 treatments were able to improve the relaxation profile in ApoE-KO mice. Short-term effects of BM-573 were mainly mediated by an increased phosphorylation of both eNOS and Akt, whereas BM-573 in vivo treatment also reduced oxidative stress and restored NO bioavailability. In addition, chronic administration of BM-573 reduced systolic blood pressure in ApoE-KO mice. In conclusion, pharmacological modulation of TxA2 biosynthesis and biological activities by dual TP antagonism/TxAS inhibition with BM-573, already known to prevent plaque formation, has the potential to correct vasodilatory dysfunction at the early stages of atherosclerosis.