[Neurology: what's new in 2022]. / Neurologie : ce qui a changé en 2022.

The year 2022 was marked by the development of numerous new treatments for refractory myasthenia gravis. The link between epilepsy and cerebrovascular disorder was studied and lamotrigine discovered to be the optimal treatment choice for epilepsy secondary to stroke to prevent mortality on patient of 45 years and older. New randomized study finally demonstrated the utility of thrombectomy in selected patients with basilar artery occlusion. The causal relationship between Epstein-Barr infection and multiple sclerosis has been proved thanks to a large cohort study. A new possibility of subcutaneous continuous levodopa administration gave promising result. Finally, numerous studies confirmed the efficacy and excellent tolerability of anti-CGRP antibodies.

L'année 2022 a été marquée par l'arrivée de nombreux traitements pour la myasthénie réfractaire. Le lien entre l'épilepsie et le risque cérébro-vasculaire a été bien étudié, démontrant que la lamotrigine semble être le meilleur traitement pour prévenir la mortalité chez les patients de 45 ans et plus. De nouvelles études ont enfin pu établir l'utilité de la thrombectomie dans les occlusions basilaires. Le lien entre le virus d'Epstein-Barr et la sclérose en plaques a pu être prouvé à la suite d'une importante étude de cohorte. Une nouvelle technique d'administration sous-cutanée de la lévodopa semble prometteuse. Enfin, de nombreuses études confirment l'efficacité et l'excellente tolérance des anticorps anti-CGRP (Calcitonine Gene Related Protein).

Laboratory Testing for the Evaluation of Phenotypic Activated Protein C Resistance.

Activated protein C (APC) resistance (APCR) is considered a risk factor of venous thromboembolism (VTE). The most common genetic disorder conferring APCR is a factor (F) V Leiden mutation, but many other factors are also implicated, such as other F5 mutations (e.g., FV Hong-Kong and FV Cambridge), protein S deficiency, elevated factor VIII, exogenous hormone use, pregnancy and postpartum, depending on how APCR is defined. Considering the large population affected, the detection of this phenotype is crucial. Two types of tests are currently available: clotting time-based assays (with several versions) and thrombin generation-based assays with the endogenous thrombin potential (ETP)-based assay. The purpose of this review is therefore to discuss the performances of these tests and the cases in which it would be appropriate to use one over the other. Initially, as APCR was thought to be solely related to the FV Leiden mutation, the objective was to obtain a 100% specific assay. Clotting-time based assays were thus specifically designed to detect this inherited condition. Later on, an APCR condition without a FV Leiden mutation was identified and highlighted as an independent risk factor of VTE. Therefore, the development of a less specific assay was needed and a global coagulation test was proposed, known as the ETP-based APCR assay. In light of the above, these tests should not be used for the same purpose. Clotting time-based assays should only be recommended as a screening test for the detection of FV mutations prior to confirmation by genetic testing. On the other hand, the ETP-based APC resistance assay, in addition to being able to detect any type of APCR, could be proposed as a global screening test as it assesses the entire coagulation process.

Laboratory Testing for the Evaluation of Phenotypic Activated Protein C Resistance.

Activated protein C (APC) resistance (APCR) is considered a risk factor of venous thromboembolism (VTE). The most common genetic disorder conferring APCR is a factor (F) V Leiden mutation, but many other factors are also implicated, such as other F5 mutations (e.g., FV Hong-Kong and FV Cambridge), protein S deficiency, elevated factor VIII, exogenous hormone use, pregnancy and postpartum, depending on how APCR is defined. Considering the large population affected, the detection of this phenotype is crucial. Two types of tests are currently available: clotting time-based assays (with several versions) and thrombin generation-based assays with the endogenous thrombin potential (ETP)-based assay. The purpose of this review is therefore to discuss the performances of these tests and the cases in which it would be appropriate to use one over the other. Initially, as APCR was thought to be solely related to the FV Leiden mutation, the objective was to obtain a 100% specific assay. Clotting-time based assays were thus specifically designed to detect this inherited condition. Later on, an APCR condition without a FV Leiden mutation was identified and highlighted as an independent risk factor of VTE. Therefore, the development of a less specific assay was needed and a global coagulation test was proposed, known as the ETP-based APCR assay. In light of the above, these tests should not be used for the same purpose. Clotting time-based assays should only be recommended as a screening test for the detection of FV mutations prior to confirmation by genetic testing. On the other hand, the ETP-based APC resistance assay, in addition to being able to detect any type of APCR, could be proposed as a global screening test as it assesses the entire coagulation process.

Oral Contraceptives and Venous Thromboembolism: Focus on Testing that May Enable Prediction and Assessment of the Risk.

Combined oral contraceptives (COCs) induce several changes in the levels of coagulation factors. The levels of procoagulant factors are often increased, while levels of anticoagulant factors are decreased. Fibrinolysis is also affected, even if the effect seems to be more counterbalanced by opposite regulation of profibrinolytic and antifibrinolytic factors. These effects on hemostasis are more pronounced with third- or fourth-generation COC compared with second-generation COC. Venous thromboembolism (VTE) risk increases when multiple risk factors, including genetic and environmental, are present simultaneously. COC use causes changes in coagulation that modify the prothrombotic state induced by preexisting hemostatic alterations in a supra-additive manner. Therefore, testing appears to be of importance not only before implementing COC but also to monitor any potential thrombogenicity induced by COC therapy. Inherited genetic factors, such as factor V Leiden, G20210A prothrombin mutation, antithrombin, protein C or protein S deficiencies, non-O blood group, as well as CYP2C9*2 and the rs4379368 mutations, have all been identified as genetic predictive risk factors of VTE in women. Nevertheless, the screening of these genetic biomarkers is not capable of assessing the phenotypic expression of the risk. This review will focus on the different options for screening the thrombogenic status in this population. Specific attention will be given to the endogenous thrombin potential-based activated protein C resistance, a test aiming at assessing the thrombogenicity induced by hormonal therapies and inherited or acquired thrombophilia.

Validation and standardization of the ETP-based activated protein C resistance test for the clinical investigation of steroid contraceptives in women: an unmet clinical and regulatory need.

Background Regulatory bodies recommend the use of an assay based on the assessment of the endogenous thrombin potential (ETP) for the investigation of the activated protein C resistance (APCr) in the development of steroid contraceptives in women. However, the assays described in the literature are home-made and not standardized regarding the method, the reagents, the reference plasma and the quality controls. In the absence of any commercially available method, we aimed at validating the ETP-based APCr assay. Methods The validation was performed according to regulatory standards. The method targets a 90% inhibition of the ETP in healthy donors in the presence of APC compared to the same condition in the absence of APC. As a large-scale production of a pool of plasma from well-selected healthy donors is impossible, algorithms were applied to a commercial reference plasma to correlate with the selected pool. Results Repeatability and intermediate precision passed the acceptance criteria. The assay demonstrated a curvilinear dose response to protein S and APC concentrations (R2 > 0.99). Analysis of plasma samples from 47 healthy individuals (22 women not taking combined hormonal contraceptives [CHC], and 25 men not Factor V Leiden carriers) confirmed the validity of the test, with a mean inhibition percentage of 90%. Investigations in 15 women taking different contraceptives and in two subjects with Factor V Leiden confirmed the good sensitivity and performance of the assay. Conclusions This validation provides the pharmaceutical industry, the regulatory bodies and physicians with a reproducible, sensitive and validated gold-standard ETP-based APCr assay.

Procoagulant activity of extracellular vesicles as a potential biomarker for risk of thrombosis and DIC in patients with acute leukaemia.

Haemostatic complication is common for patients with hematologic malignancies. Recent studies suggest that the procoagulant activity (PCA) of extracellular vesicles (EV) may play a major role in venous thromboembolism and disseminated intravascular coagulation (DIC) in acute leukaemia. To study the impact of EVs from leukaemic patients on thrombin generation and to assess EV-PCA as a potential biomarker for thrombotic complications in patients with acute leukaemia. Blood samples from a cohort of patients with newly diagnosed acute leukaemia were obtained before treatment (D-0), 3 and 7 days after treatment (D-3 and D-7). Extracellular vesicles were isolated and concentrated by ultracentrifugation. EV-PCA was assessed by thrombin generation assay, and EV-associated tissue factor activity was measured using a commercial bio-immunoassay (Zymuphen MP-TF®). Of the 53 patients, 6 had increased EV-PCA at D-0 and 4 had a thrombotic event. Patients without thrombotic events (n = 47) had no elevated EV-PCA. One patient had increased EVs with procoagulant activity at D-3 and developed a DIC at D-5. This patient had no increased EVs-related tissue factor activity from D-0 to D-7 (<2 pg/ml). Eight patients had increased EVs with tissue factor activity (>2 pg/ml), of these, four had a thrombosis and two had haemorrhages. Procoagulant activity of extracellular vesicles could have a predictive value in excluding the risk of thrombotic events. Our findings also suggest a possible association between thrombotic events and EV-PCA.

Evaluation of Activated Protein C Resistance Using Thrombin Generation Test.

Activated protein C (APC) resistance (APCR) has been identified as a risk factor of venous thromboembolism (VTE). A mutation at the level of factor (F) V has at first permitted the description of this phenotypic pattern and corresponded to a transition (guanine to adenine) at nucleotide 1691 in the gene coding for factor V, resulting in the replacement of arginine at position 506 by a glutamine. This confers to this mutated FV a resistance toward the proteolytic action of the complex formed by activated protein C with protein S. However, many other factors also lead to APCR, such as other F5 mutations (e.g., FV Hong Kong and FV Cambridge), protein S deficiency, elevated factor VIII, exogenous hormone use, pregnancy, and postpartum. All these conditions lead to the phenotypic expression of APCR and are associated with an increased risk of VTE. Considering the large population affected, the proper detection of this phenotype is a public health challenge. Currently, two types of tests are available: clotting time-based assays and their multiple variants and a thrombin generation-based assays and the endogenous thrombin potential (ETP)-based APCR assay. As APCR was thought to be uniquely related to the FV Leiden mutation, clotting time-based assays were specifically designed to detect this inherited condition. Nevertheless, other APCR conditions have been reported but were not captured by these clotting methods. Thus, the ETP-based APCR assay has been proposed as a global coagulation test able to these multiple APCR conditions, as it provides much more information, which makes it a potential candidate for screening coagulopathic conditions before therapeutic interventions. This chapter will describe the current method used for the realization of the ETP-based APC resistance assay.

The edoxaban-M4 metabolite and measurement of edoxaban by chromogenic assays in human plasma.

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.

Comparison of analytical performances between clot waveform analysis and FibWave in edoxaban-treated patients and healthy controls.

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.

NETosis and Nucleosome Biomarkers in Septic Shock and Critical COVID-19 Patients: An Observational Study.

Background: Neutrophil extracellular traps' (NETs') formation is a mechanism of defense that neutrophils deploy as an alternative to phagocytosis, to constrain the spread of microorganisms. Aim: The aim was to evaluate biomarkers of NETs' formation in a patient cohort admitted to intensive care unit (ICU) due to infection. Methods: Forty-six septic shock patients, 22 critical COVID-19 patients and 48 matched control subjects were recruited. Intact nucleosomes containing histone 3.1 (Nu.H3.1), or citrullinated histone H3R8 (Nu.Cit-H3R8), free citrullinated histone (Cit-H3), neutrophil elastase (NE) and myeloperoxidase (MPO) were measured. Results: Significant differences in Nu.H3.1 and NE levels were observed between septic shock and critical COVID-19 subjects as well as with controls (p-values < 0.05). The normalization of nucleosome levels according to the neutrophil count improved the discrimination between septic shock and critical COVID-19 patients. The ratio of Nu.Cit-H3R8 to Nu.H3.1 allowed the determination of nucleosome citrullination degree, presumably by PAD4. Conclusions: H3.1 and Cit-H3R8 nucleosomes appear to be interesting markers of global cell death and neutrophil activation when combined. Nu.H3.1 permits the evaluation of disease severity and differs between septic shock and critical COVID-19 patients, reflecting two distinct potential pathological processes in these conditions.

Interlaboratory variability of activated protein C resistance using the ETP-based APC resistance assay.

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.

Impact of centrifugation on thrombin generation in healthy subjects and in patients treated with direct oral anticoagulants.

INTRODUCTION: Double centrifugation before freezing is recommended before thrombin generation assays (TGA). However, this procedure is not mandatory for routine hemostasis tests, precluding the use of these samples for TGA. The aim of this study is to assess the impact of single and double centrifugation on TGA performed on frozen samples from healthy volunteers (HVs) and patients receiving direct oral anticoagulants (DOACs). METHODS: Forty HVs and 57 patients receiving a DOAC (dabigatran, rivaroxaban, apixaban, or edoxaban) were included in this prospective double-center observational study. Blood was collected into 109 mmol/L citrated tubes and frozen at -70°C before TGA using ST Genesia with STG-DrugScreen reagent. Four pre-analytical conditions were studied: (A) single centrifugation (2000 g, 15 minutes) before freezing; (B) one centrifugation before freezing and another after thawing (2000 g, 15 minutes for both); (C) one centrifugation before freezing(2000 g, 15 minutes) and another after thawing (2000 g, 10 minutes); (D) double centrifugation (2000 g, 15 minutes) before freezing (reference). Centrifugation conditions (A), (B), and (C) were compared with the reference condition (D). Acceptable relative differences were defined at 6%, 8%, and 10% for normalized lag time, endogenous thrombin potential, and peak height, respectively. RESULTS: Centrifugation conditions had a small but acceptable impact on HVs samples, but single centrifugation always resulted in unacceptable reductions in normalized lag times for DOAC samples. A second centrifugation after thawing permitted the recovery of acceptable differences for the three TGA parameters for edoxaban but not for apixaban, rivaroxaban, nor dabigatran. CONCLUSION: Double centrifugation before freezing should remain the recommended pre-analytical condition before TGA.

Proof of concept of a new scale for the harmonization and the standardization of the ETP-based APC resistance.

BACKGROUND: The evaluation of the activated protein C resistance (APCr) based on the endogenous thrombin potential (ETP) is recommended during the development of steroid contraceptives. Results are usually expressed as "normalized APC sensitivity ratio" (nAPCsr) using a reference plasma that should achieve an ETP ratio of 0.1 in presence of exogenous APC. Because of the interassay variability, achieving exactly an ETP ratio of 0.1 in each run is almost impossible, which significantly affects the theoretical 0-10 scale of nAPCsr. OBJECTIVES: To compare the nAPCsr to the nAPCsr10 , a newly proposed method to express the degree of APC resistance. METHODS: Individual plasma samples (n = 854) were analyzed to compare nAPCsr and nAPCsr10 . These values were obtained using the validated ETP-based APCr assay. RESULTS: The Spearman correlation between nAPCsr and nAPCsr10 had a coefficient of 0.99. Linear regression showed the following equation y = 0.9315*x + 0.03942 (r2  = .97). When differences (nAPCsr10 - nAPCsr) were plotted against nAPCsr10 , the mean difference equaled 0.16% or 4.95%. The correction obtained with the use of the nAPCsr10 showed that the results of the nAPCsr were statistically different (P < .0001). CONCLUSIONS: This new scale provides a harmonization and normalization of the nAPCsr. Results show a better reproducibility with the nAPCsr10 . It avoids the additional variability and the unharmonized scale introduced by the use of a reference plasma. This adapted method for the calculation of the APC resistance could provide the regulatory and scientific bodies with more reproducible and harmonized evaluations.

The Risk of Arterial Thrombosis in Patients With Chronic Myeloid Leukemia Treated With Second and Third Generation BCR-ABL Tyrosine Kinase Inhibitors May Be Explained by Their Impact on Endothelial Cells: An In-Vitro Study.

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.

Development of new methodologies for the chromogenic estimation of betrixaban concentrations in plasma.

INTRODUCTION: Chromogenic anti-Xa assays are the most appropriate tests to estimate the amount of betrixaban in plasma but the sensitivity of available tests is limited and improvements are needed to encompass the on-therapy range. METHODS: Betrixaban was spiked at concentrations ranging from 0 to 500 ng/mL in plasma from healthy donors. Three commercial tests were used (Biophen® DiXaI® , STA® Liquid Anti-Xa, and HemosIL® Liquid Anti-Xa), and adaptation of their sample dilution scheme was performed. These new methodologies were also tested on plasma spiked with amounts of unfractionated heparin (UFH), low molecular weight heparins (LMWH), or fondaparinux covering the on-therapy ranges to evaluate their sensitivity to indirect factor Xa inhibitors. RESULTS: Results showed concentration-dependent decreases in OD/min inversely proportional to the dilutions. While modifications improve the sensitivity of these tests to betrixaban (eg, ½*OD/min of 502 ng/mL [95% CI: 495-508 ng/mL] for Biophen® DiXaI® [1:50] is reduced to 51 ng/mL [95% CI: 50-52 ng/mL] for improved Biophen® DiXaI® [1:5]), results also showed an increased sensitivity to indirect factor Xa inhibitors, except for Biophen® DiXaI® which remains insensitive to UFH and LMWH. CONCLUSIONS: Results showed that the improvement of current chromogenic anti-Xa methodologies enhances the sensitivity of these assays to betrixaban but also to indirect factor Xa inhibitors. This lack of specificity may lead to overestimation of betrixaban concentrations in patients bridged with heparins. To avoid this cross-interference, the use of the Biophen® DiXaI® may be a solution except for fondaparinux which remains active even in the presence of the Biophen® DiXaI® 's specific buffer. For the other chromogenic assays, the conception and validation of specific buffer is required.

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.

Optimal wavelength for the clot waveform analysis: Determination of the best resolution with minimal interference of the reagents.

INTRODUCTION: Clot waveform analysis (CWA), a new methodology to assess coagulation process, can be usefully applied in various clinical settings. However, its clinical use is limited mainly because of the absence of standardization. No consensus exists regarding the wavelengths at which CWA has to be performed what is crucial for the sensitivity of the CWA. OBJECTIVES: The primary aim of this study is to determine which wavelength is the most sensitive and specific for CWA. Interindividual baseline absorbance will also be assessed as the impact of reagents from the intrinsic, extrinsic, and common coagulation pathway will be determined. METHODS: Plasma samples were screened at wavelengths from 280 to 700 nm to provide absorbance spectra in clotted and nonclotted plasma. The interindividual variability of baseline absorbance was obtained by screening plasma from 50 healthy individuals at 340, 635, and 671 nm. The inner-filter effect of reagents was assessed in plasma or serum when appropriate at the same wavelengths. The reagents were those commonly used for activated partial thromboplastin time, prothrombin time, thrombin time, and dilute Russell's viper venom time. RESULTS: Clotted plasma has higher absorbance value than nonclotted plasma (P < 0.01). The absorbance of all type of samples is higher at 340 nm than at >600 nm (P < 0.01). The interindividual variability at the different wavelengths was around 25%. However, except with the STA®-CKPrest® and STA®-NeoPTimal®, the reagents do not have a significant effect on the baseline absorbance. CONCLUSIONS: Wavelengths above 650 nm are recommended to perform CWA. Most of the commercialized reagents can be used for CWA.

Circulating MicroRNAs as Biomarkers in Diffuse Large B-cell Lymphoma: A Pilot Prospective Longitudinal Clinical Study.

OBJECTIVES: Diffuse large B-cell lymphoma (DLBCL) is highly heterogeneous in terms of phenotype and treatment response in patients. These characteristics make the prognosis difficult to establish and hinder the use of new personalized treatments in clinical practice. In this context, there is currently a need to define new biomarkers enabling a better definition of DLBCL subtypes, prognosis evaluation, and an overview of the resistance to chemotherapeutics. The aim of this study was to evaluate the use of microRNAs found in plasma from patients with DLBCL as biomarkers of tumor evolution in these patients. METHOD: For this purpose, a plasma biobank was created with samples from patients with DLBCL. The evolution of the level of selected microRNAs during treatment has been studied. A total of 19 patients with DLBCL were included in this pilot mono-centered study and a total of 68 samples were analyzed. RESULTS: The first step of this study was the selection of the microRNAs to be quantified in all the samples of the biobank and that could potentially be used as biomarkers. To this end, quantification of 377 microRNAs was performed on the plasma samples of 2 selected patients with DLBCL and 1 healthy donor with no history of cancer. Among the 377 microRNAs evaluated, 7 were selected and analyzed in the entire biobank. CONCLUSIONS: This study highlighted 5 circulating microRNAs whose plasma levels would be worth further investigating for the characterization of DLBCL evolution in patients. MiR-21 and miR-197 had a significant higher plasmatic level in patients with tumors unresponsive to treatment. With a higher plasma level in patients with complete remission, miR-19b, miR-20a, and miR-451 could enable to differentiate, at the remission review, patients with residual tumor, from patients with complete remission.

Locus Coeruleus atrophy doesn't relate to fatigue in Parkinson's disease.

Fatigue is a frequent complaint among healthy population and one of the earliest and most debilitating symptoms in Parkinson's disease (PD). Earlier studies have examined the role of dopamine and serotonin in pathogenesis of fatigue, but the plausible role of noradrenalin (NA) remains underexplored. We investigated the relationship between fatigue in Parkinsonian patients and the extent of degeneration of Locus Coeruleus (LC), the main source of NA in the brain. We quantified LC and Substantia Nigra (SN) atrophy using neuromelanin-sensitive imaging, analyzed with a novel, fully automated algorithm. We also assessed patients' fatigue, depression, sleep disturbance and vigilance. We found that LC degeneration correlated with the levels of depression and vigilance but not with fatigue, while fatigue correlated weakly with atrophy of SN. These results indicate that LC degeneration in Parkinson's disease is unlikely to cause fatigue, but may be involved in mood and vigilance alterations.

Transfer of multidrug resistance among acute myeloid leukemia cells via extracellular vesicles and their microRNA cargo.

The treatment of acute leukemia is still challenging due in part to the development of resistance and relapse. This chemotherapeutics resistance is established by clonal selection of resistant variants of the cancer cells. Recently, a horizontal transfer of chemo-resistance among cancer cells via extracellular vesicles (EVs) has been suggested. The aim of this research was to investigate the role of EVs in chemo-resistance in acute myeloid leukemia. For this purpose, the sensitive strain of the promyelocytic leukemia HL60 cell line was studied along with its multi-resistant strain, HL60/AR that overexpresses the multidrug resistance protein 1 (MRP-1). A chemo-resistance transfer between the two strains was established by treating HL60 cells with EVs generated by HL60/AR. This study reveals that EVs from HL60/AR can interact with HL60 cells and transfer at least partially, their chemo-resistance. EVs-treated cells begin to express MRP-1 probably due to a direct transfer of MRP-1 and nucleic acids transported by EVs. In this context, two microRNAs were highlighted for their high differential expression in EVs related to sensitive or chemo-resistant cells: miR-19b and miR-20a. Because circulating microRNAs are found in all biological fluids, these results bring out their potential clinical use as chemo-resistance biomarkers in acute myeloid leukemia.