Prospective and comparative study of paroxysmal nocturnal hemoglobinuria patients treated or not by eculizumab: Focus on platelet extracellular vesicles.

Thrombosis are severe complications of paroxysmal nocturnal hemoglobinuria (PNH), effectively reduced by eculizumab. Extracellular vesicles (EVs) may play a central role. The objective of this study was to assess the procoagulant activity of plasma isolated from PNH patients (treated or not by eculizumab) and to quantify their circulating EVs.We iteratively collected the platelet-free-plasma of 17 PNH patients and 16 matched healthy volunteers, quantified their circulating EVs by flow cytometry and evaluated their procoagulant activity by thrombin generation and STA-Procoag-procoagulant phospholipid (PPL) assays.A significant decrease of EVs from platelets (P = .024) and an increase of the STA-Procoag-PPL clotting time (P = .049) was observed after initiation of eculizumab and up to 11 weeks after. This reduction of prothrombotic biomarkers was not observed with the thrombin generation test due to a lack of sensitivity of this assay. Active hemolysis was observed in 90% of patients and elevated D-dimers in 41% of them. However, no significant difference was observed between patients and control subjects regarding the procoagulant activity, the EVs quantity, or the cellular origin. Lactate dehydrogenase (LDH) levels were lower in eculizumab-treated patients compared to nontreated patients (441 vs 2448 IU/L). D-dimers and LDH decreased after administration of eculizumab (mean decrease of 1307 ng/mL and 4159 IU/L, respectively).These observations suggest a decrease of the phospholipid-dependent procoagulant potential of EVs after eculizumab therapy in PNH patients. TRIAL REGISTRATION:: NUB: B039201214365.

Assessment of the analytical performances and sample stability on ST Genesia system using the STG-DrugScreen application.

BACKGROUND: Thrombin generation testing has been used to provide information on the coagulation phenotype of patients. The most used technique is the calibrated automated thrombogram (CAT) but it suffers from a lack of standardization, preventing its implementation in routine. The ST Genesia is a new analyzer designed to assess thrombin generation based on the same principle as the CAT. Unlike the CAT system, the ST Genesia is a benchtop, fully automated analyzer, able to perform the analyses individually and not by batch, with strict control of variables such as temperature and volumes, ensuring, theoretically, maximal reproducibility. OBJECTIVES: This study aimed at assessing the performance of the STG-DrugScreen application on the ST Genesia analyzer. We also aimed at exploring stability of plasma samples after freezing and defining a reference normal range. RESULTS: Results demonstrated the excellent interexperiment precision of the ST Genesia and confirmed that the use of a reference plasma helps reducing the inter-experiments variability. Stability revealed that plasma samples are stable for at least 11 months at -70°C or lower, except for those containing low molecular weight heparins which have to be tested within 6 months. Freezing had no effect on the majority of thrombin generation parameters except on time to peak. CONCLUSIONS: Our results suggest an easy implementation of thrombin generation with the use of ST Genesia in the routine laboratory. This will facilitate the design of multicentric studies and enable the establishment of reliable and evidence-based thresholds, which may improve the management of patients treated with anticoagulants.

Extracellular Vesicles in Red Blood Cell Concentrates: An Overview.

Red blood cell (RBC) concentrates may be stored for up to 42 days before transfusion to a patient. During storage extracellular vesicles (EVs) develop and can be detected in significant amounts in RBC concentrates. The concentration of EVs is affected by component preparation methods, storage solutions, and inter-donor variation. Laboratory investigations have focused on the effect of EVs on in vitro assays of thrombin generation and immune responses. Assays for EVs in RBC concentrates are not standardized. The aims of this review are to describe the factors that determine the presence of erythrocyte-EVs in RBC concentrates, the current techniques used to characterize them, and the potential role of EV analysis as a quality control maker for RBC storage.

Diagnosis and management of PNH: Review and recommendations from a Belgian expert panel.

Despite its considerable morbidity and mortality, paroxysmal nocturnal haemoglobinuria (PNH) is still underdiagnosed. Patients with PNH can suffer from cardiovascular, gastrointestinal, neurological or haematological symptoms and refer to several specialists. The aim of this paper is to review the diagnosis and the management of PNH patients, with the primary focus on identifying high-risk groups. Additionally, the implementation and prognostic value of the defined high-risk groups will be commented on and the management of PNH patients is discussed from a Belgian perspective. Finally, based on the available data, recommendations are provided. Eculizumab is a potent C5 complement inhibitor and reduces intravascular haemolysis and thrombosis in PNH patients and improves their quality of life. As thrombosis is the main cause of death in PNH patients, identifying high-risk PNH patients in need of therapy is essential. Currently, novel complement inhibitors are in development and the first data seem promising. Another challenge in PNH is to identify new markers to assess the thrombotic risk to achieve a better risk-based prophylactic anti-thrombotic management. Finally, because of the low prevalence of the disease, PNH patients should be included in the prospective PNH registry, which will offer new insights on the natural course of the disease and the impact of treatment of PNH.

Preventability of serious thromboembolic and bleeding events related to the use of oral anticoagulants: a prospective study.

AIMS: To determine the preventability of serious adverse drug reactions (ADRs) related to the use of direct oral anticoagulants (DOACs), and to explore contributing factors to preventable ADRs. Results were compared with vitamin K antagonists (VKAs). METHODS: We conducted a prospective observational study in the emergency departments of two teaching hospitals from July 2015 to January 2016. Patients admitted with a thrombotic or bleeding event while under DOAC or VKA were included. Four independent reviewers assessed causality, seriousness and preventability of ADRs using pilot-tested scales. For cases of serious and potentially preventable ADRs, we performed semi-structured interviews with general practitioners to identify contributing factors to ADRs. The primary outcome was the proportion of serious ADRs that were potentially preventable. RESULTS: The analysis included 46 DOAC and 43 VKA patients (median age 79 years). Gastrointestinal (n = 34) and intracranial (n = 16) bleedings were the most frequent ADRs. Results were that 53% of DOAC- and 61% of VKA-related serious ADRs were deemed potentially preventable. Prescribing issues and inadequate monitoring were frequent for DOAC and VKA respectively. We identified many causes of preventable ADRs that applied to all oral anticoagulants, such as pharmacodynamic drug interactions and lack of communication. CONCLUSIONS: More than half of serious ADRs were potentially preventable for both DOACs and VKAs. Interventions focusing on prescribing, patient education and continuity of care should help improve the use of DOACs in practice.

Evaluation of the DOAC-Stop® Procedure to Overcome the Effect of DOACs on Several Thrombophilia Screening Tests.

The impact of direct oral anticoagulants (DOACs) on laboratory assays used for thrombophilia testing (e.g., antithrombin, protein S, protein C, lupus anticoagulant and activated protein-C resistance) is a well-known issue and may cause false-positive and -negative results. Therefore, the correct interpretation of tests that are performed in patients taking DOACs is mandatory to prevent misclassification and the subsequent clinical consequences. We aimed at evaluating the efficiency of a new and simple procedure (DOAC-Stop®; Haematex Research, Hornsby, Australia) to overcome the effect of all DOACs in real-life settings and to assess the percentage of erroneous results due to the presence of DOACs on thrombophilia screening tests. For this purpose, 135 DOAC-treated patients (38 apixaban, 40 dabigatran, 15 edoxaban, and 42 rivaroxaban) and 20 control patients were enrolled. A significant drop in apixaban, dabigatran, edoxaban, and rivaroxaban plasma concentrations following the DOAC-Stop® treatment was observed (74.8-8.2 ng/mL [ p < 0.0001], 95.9-4.7 ng/mL [ p < 0.0001], 102.1-8.8 ng/mL [ p = 0.001], and 111.3-7.0 ng/mL [ p < 0.0001], respectively). The DOAC-Stop® treatment was mostly effective to overcome the effect of DOACs on PTT-LA, dilute Russell's viper venom time (dRVVT) screen, and dRVVT confirm tests. Using our procedures, false-positive results due to DOACs were observed only with lupus anticoagulant tests (up to 75%) and fell to zero after the DOAC-Stop® procedure, regardless of the DOAC considered. In conclusion, the DOAC-Stop® adsorbent procedure appeared to be an effective and simple way to overcome the interference of DOAC on coagulation tests and should facilitate the interpretation of thrombophilia screening tests in patients taking DOACs.

Application of a clot-based assay to measure the procoagulant activity of stored allogeneic red blood cell concentrates.

BACKGROUND: Thrombotic effects are possible complications of red blood cell transfusion. The generation and accumulation of procoagulant red blood cell extracellular vesicles during storage may play an important role in these thrombotic effects. The objective of this study was to assess the value of a simple phospholipid-dependent clot-based assay (STA®-Procoag-PPL) to estimate the procoagulant activity of stored red blood cells and changes in this activity during storage of the blood component. MATERIALS AND METHODS: Extracellular vesicles from 12 red blood cell concentrates were isolated at 13 storage time-points and characterised by quantitative and functional methods: the degree of haemolysis (direct spectrophotometry), the quantification and determination of cellular origin (flow cytometry) and the procoagulant activity (thrombin generation and STA®-Procoag-PPL assays) were assessed. RESULTS: The mean clotting time of extracellular vesicles isolated from red blood cell concentrates decreased from 117.2±3.6 sec on the day of collection to 33.8±1.3 sec at the end of the storage period. This illustrates the phospholipid-dependent procoagulant activity of these extracellular vesicles, as confirmed by thrombin generation. Results of the peak of thrombin and the STA®-Procoag-PPL were well correlated (partial r=-0.41. p<0.001). In parallel, an exponential increase of the number of red blood cell-derived extracellular vesicles from 1,779/µL to 218,451/µL was observed. DISCUSSION: The STA®-Procoag-PPL is a potentially useful technique for assessing the procoagulant activity of a red blood cell concentrate.

Eculizumab decreases the procoagulant activity of extracellular vesicles in paroxysmal nocturnal hemoglobinuria: A pilot prospective longitudinal clinical study.

INTRODUCTION: Paroxysmal nocturnal hemoglobinuria (PNH) is a disease characterized by the susceptibility of blood cells to attack by the complement system, inducing extracellular vesicle (EV) production. Thromboembolism is the leading cause of death in this condition. Eculizumab, a humanized monoclonal antibody which inhibits the C5 protein of the complement, reduces the thrombotic risk in PNH. MATERIALS AND METHOD: We conducted a pilot, prospective, open-label, longitudinal clinical study with six PNH patients treated with eculizumab. The aim was to measure, by flow cytometry, the EVs' production in the patients' platelet-free plasma (PFP) before and during the treatment. We also assessed the procoagulant activity in PFP using STA®-Procoag-PPL and thrombin generation assays (TGA). A high-sensitive version of TGA was also used to study the procoagulant profile induced by the EVs using EVs pelleted from PFP. RESULTS: We observed a decrease in platelet EV count with eculizumab treatment (p<0.05). STA®-Procoag-PPL assay showed a decrease of the procoagulant profile induced by procoagulant phospholipids (PL) during treatment. These results were not confirmed by TGA on PFP, due to a lack of sensitivity. Thus, we used a high-sensitive version of TGA that enabled us to observe variation in the procoagulant profile induced by the EVs with eculizumab (p<0.05). CONCLUSIONS: Eculizumab has an impact on the extent of EV production and on the procoagulant profile induced by the procoagulant PL and the EVs. One factor in the antithrombotic action of eculizumab is its ability to decrease EV production and the procoagulant profile induced by PL and EVs.

Tips and tricks for flow cytometry-based analysis and counting of microparticles.

Submicron-sized extra-cellular vesicles generated by budding from the external cell membranes, microparticles (MPs) are important actors in transfusion as well as in other medical specialties. After briefly positioning their role in the characterization of labile blood products, this technically oriented chapter aims to review practical points that need to be considered when trying to use flow cytometry for the analysis, characterization and absolute counting of MP subsets. Subjects of active discussions relative to instrumentation will include the choice of the trigger parameter, possible standardization approaches requiring instrument quality-control, origin and control of non-specific background and of coincidence artifacts, choice of the type of electronic signals, optimal sheath fluid and sample speed. Questions related to reagents will cover target antigens and receptors, multi-color reagents, negative controls, enumeration of MPs and limiting artifacts due to unexpected (micro-) coagulation of plasma samples. Newly detected problems are generating innovative solutions and flow cytometry will continue to remain the technology of choice for the analysis of MPs, in the domain of transfusion as well as in many diverse specialties.

Non-VKA Oral Anticoagulants: Accurate Measurement of Plasma Drug Concentrations.

Non-VKA oral anticoagulants (NOACs) have now widely reached the lucrative market of anticoagulation. While the marketing authorization holders claimed that no routine monitoring is required and that these compounds can be given at fixed doses, several evidences arisen from the literature tend to demonstrate the opposite. New data suggests that an assessment of the response at the individual level could improve the benefit-risk ratio of at least dabigatran. Information regarding the association of rivaroxaban and apixaban exposure and the bleeding risk is available in the drug approval package on the FDA website. These reviews suggest that accumulation of these compounds increases the risk of experiencing a bleeding complication. Therefore, in certain patient populations such as patients with acute or chronic renal impairment or with multiple drug interactions, measurement of drug exposure may be useful to ensure an optimal treatment response. More specific circumstances such as patients experiencing a haemorrhagic or thromboembolic event during the treatment duration, patients who require urgent surgery or an invasive procedure, or patient with a suspected overdose could benefit from such a measurement. This paper aims at providing guidance on how to best estimate the intensity of anticoagulation using laboratory assays in daily practice.

Does the Russell Viper Venom time test provide a rapid estimation of the intensity of oral anticoagulation? A cohort study.

BACKGROUND: Dilute Russell Viper Venom Time (DRVV-T) might be useful in urgent settings for screening patients on Non-VKA Oral Anticoagulants (NOACs). AIM: To compare the accuracy of DRVV-T with gold standard assays for the assessment of pharmacodynamics of dabigatran, rivaroxaban and vitamin K antagonist (VKA) in plasma samples from patients. METHODS: Sixty rivaroxaban, 48 dabigatran and 50 VKA samples from patients were included. DRVV-T was performed in all groups using STA®-Staclot®DRVV-Screen and -Confirm. For NOACs, PT and aPTT were performed using different reagents while plasma drug concentrations were measured by liquid mass-spectrometry (LC-MS/MS). For VKA, INR was performed using RecombiPlasTin 2G®. RESULTS: For NOACs, correlations between calibrated STA®-Staclot®DRVV-Confirm and LC-MS/MS (rs=0.88 and 0.97 for rivaroxaban and dabigatran, respectively) were higher than the ones obtained with STA®-Staclot®DRVV-Screen (rs=0.87 and 0.91), PT (rs=0.83 to 0.86) or aPTT (rs=0.84 to 0.89). Bland Altman analyses showed that calibrated DRVV-T methods tend to overestimate plasma concentrations of NOACs. ROC curves revealed that cut-off to exclude supra-therapeutic levels at Ctrough (i.e. 200ng/mL) are different for dabigatran and rivaroxaban. Neither STA®-Staclot®DRVV-Screen nor -Confirm correlated sufficiently with the intensity of VKA therapy (rs=0.35 and 0.52). CONCLUSIONS: STA®-Staclot®DRVV-Confirm provides a rapid estimation of the intensity of anticoagulation with rivaroxaban or dabigatran without specific calibrators. At Ctrough, thresholds for rivaroxaban and dabigatran can be used to identify supra-therapeutic plasma level. However, this test cannot differentiate the nature of the NOACs. The development of a point-of-care device optimising this method would be of particular interest in emergency situations.

Pathophysiology, diagnosis, and treatment of paroxysmal nocturnal hemoglobinuria: a review.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired disorder of the hematopoietic stem cell that makes blood cells more sensitive to the action of complement. Patients experience intravascular hemolysis, smooth muscle dystonia, renal failure, arterial and pulmonary hypertension, recurrent infectious diseases and an increased risk of notably dreadful thrombotic complications. The diagnosis is made by flow cytometry. Efforts have been recently performed to improve the sensitivity and the standardization of this technique. PNH is frequently associated with aplastic anemia or low-risk myelodysplasia and may be asymptomatic. Management of the classical form of PNH has been dramatically revolutionized by the development of eculizumab, which brings benefits in terms of hemolysis, quality of life, renal function, thrombotic risk, and life expectancy. Prophylaxis and treatment of arterial and venous thrombosis currently remain a challenge in PNH.

Preventive strategies against bleeding due to nonvitamin K antagonist oral anticoagulants.

Dabigatran etexilate (DE), rivaroxaban, and apixaban are nonvitamin K antagonist oral anticoagulants (NOACs) that have been compared in clinical trials with existing anticoagulants (warfarin and enoxaparin) in several indications for the prevention and treatment of thrombotic events. All NOACs presented bleeding events despite a careful selection and control of patients. Compared with warfarin, NOACs had a decreased risk of intracranial hemorrhage, and apixaban and DE (110 mg BID) had a decreased risk of major bleeding from any site. Rivaroxaban and DE showed an increased risk of major gastrointestinal bleeding compared with warfarin. Developing strategies to minimize the risk of bleeding is essential, as major bleedings are reported in clinical practice and specific antidotes are currently not available. In this paper, the following preventive approaches are reviewed: improvement of appropriate prescription, identification of modifiable bleeding risk factors, tailoring NOAC's dose, dealing with a missed dose as well as adhesion to switching, bridging and anesthetic procedures.

Appropriateness of prescribing dabigatran etexilate and rivaroxaban in patients with nonvalvular atrial fibrillation: a prospective study.

BACKGROUND: Direct oral anticoagulants have been developed to address some of the drawbacks of vitamin-K antagonists. However, special attention should be given when using these drugs, especially in patients with renal insufficiency, questionable compliance, and those at high risk of bleeding. OBJECTIVE: To evaluate the appropriateness of prescribing dabigatran etexilate (DE) and rivaroxaban in patients with nonvalvular atrial fibrillation (NVAF) in real-life clinical practice. METHODS: This was a prospective study that included patients presenting to a teaching hospital from April to mid-October 2013, who were taking rivaroxaban or DE for NVAF. Appropriateness of prescribing was evaluated using 9 of the 10 criteria of the Medication Appropriateness Index. The primary outcome measure was the prevalence of inappropriate prescribing. Secondary outcome measures included (a) categories of inappropriateness, (b) prevalence of adverse drug events, and (c) interventions made by a clinical pharmacist to optimize prescribing. RESULTS: A total of 69 patients were evaluated; 16 patients (23%) had 1 inappropriate criterion, and an additional 18 (26%) had more than 1 inappropriate criterion. The most frequent inappropriate criteria were inappropriate choice (28% of patients), wrong dosage (26%), and impractical modalities of administration (26%). An adverse event (AE) was found in 51% of patients (including 8 patients with transient ischemic attack/stroke). The clinical pharmacists performed 48 interventions, and 94% were accepted by the physician. CONCLUSIONS: Inappropriate use of DE and rivaroxaban in patients with NVAF is frequent and possibly leads to AEs. Reinforcing education of health care professionals and patients is needed. Collaboration with clinical pharmacists can contribute to better use.

The central role of extracellular vesicles in the mechanisms of thrombosis in paroxysmal nocturnal haemoglobinuria: a review.

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.

[Why, when and how to monitor new oral anticoagulants]. / Pourquoi, quand et comment doser les nouveaux anticoagulants oraux?

Several direct oral anticoagulants (DOACs) are now widely used in the prevention and treatment of thromboembolic events. Unlike vitamin K antagonists, DOACs exhibit predictable pharmacokinetics and pharmacodynamics. DOACs are to be administered at fixed doses without routine coagulation monitoring. However, in some patient populations or specific clinical circumstances, measurement of drug exposure may be useful, such as in suspected overdose, in patients with a haemorrhagic or thromboembolic event during treatment with an anticoagulant, in those with acute renal failure, or in patients who require urgent surgery. This article provides practical guidance on laboratory testing of DOACs in routine practice and summarizes the influence of DOACs on commonly used coagulation assays.

Microparticle bearing tissue factor: a link between promyelocytic cells and hypercoagulable state.

Patients with hematological malignancies have a 28-fold increased risk of venous thromboembolism (VTE). Among patients with acute myelogenous leukemia (AML), the 2-year cumulative incidence of VTE is 5.2%. Several studies suggest that microvesicles (MVs) harboring TF may play a role in VTE and disseminated intravascular coagulation (DIC) in acute promyelocytic leukemia (APL). The aim of this study was to assess the capacity of untreated (APL) cells to shed procoagulant MVs. APL cells (NB4 and HL-60 cell lines) and MVs were separated by filtration (0.1-0.22-0.45-0.65 µm). The procoagulant activity (PCA) was assessed by thrombin generation assay (TGA). Alternatively, MVs were incubated with anti-Tissue Factor (TF) antibodies, with annexin V to assess the contribution of TF and phospholipids (PL) to the PCA, respectively. NB4 cells had a high PCA mainly triggered by MVs of size under 0.45 µm. The PCA of MVs was related to the expression of active TF and PL. HL-60 cells had a weaker PCA since TF is mostly present in its inactive form. Moreover, HL-60 do not produce MVs<0.65 µm associated with PCA. MVs could have a predicting value for VTE and DIC in patients with acute promyelocytic leukemia and could inform physicians about the optimal use of a thromboprophylaxis.