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Minet V.,University of Namur | Baudar J.,Namur Research Institute for LIfe science NARILIS | Bailly N.,Namur Research Institute for LIfe science NARILIS | Douxfils J.,University of Namur | And 8 more authors.
Thrombosis Research | Year: 2014

Background Accurate diagnosis of heparin-induced thrombocytopenia (HIT) is essential but remains challenging. We have previously demonstrated, in a retrospective study, the usefulness of the combination of the 4Ts score, AcuStar HIT and heparin-induced multiple electrode aggregometry (HIMEA) with optimized thresholds. Objectives We aimed at exploring prospectively the performances of our optimized diagnostic algorithm on suspected HIT patients. The secondary objective is to evaluate performances of AcuStar HIT-Ab (PF4-H) in comparison with the clinical outcome. Methods 116 inpatients with clinically suspected immune HIT were included. Our optimized diagnostic algorithm was applied to each patient. Sensitivity, specificity, negative predictive value (NPV), positive predictive value (PPV) of the overall diagnostic strategy as well as AcuStar HIT-Ab (at manufacturer's thresholds and at our thresholds) were calculated using clinical diagnosis as the reference. Results Among 116 patients, 2 patients had clinically-diagnosed HIT. These 2 patients were positive on AcuStar HIT-Ab, AcuStar HIT-IgG and HIMEA. Using our optimized algorithm, all patients were correctly diagnosed. AcuStar HIT-Ab at our cut-off (> 9.41 U/mL) and at manufacturer's cut-off (> 1.00 U/mL) showed both a sensitivity of 100.0% and a specificity of 99.1% and 90.4%, respectively. Conclusion The combination of the 4Ts score, the HemosIL® AcuStar HIT and HIMEA with optimized thresholds may be useful for the rapid and accurate exclusion of the diagnosis of immune HIT. © 2014 Elsevier Ltd. All rights reserved.


Mullier F.,Hematology Laboratory NARILIS | Mullier F.,University of Namur | Mullier F.,Namur Thrombosis and Hemostasis Center | Minet V.,University of Namur | And 12 more authors.
Thrombosis Research | Year: 2014

Background Early diagnosis of immune heparin-induced thrombocytopenia (HIT) is essential to improve clinical outcome but remains challenging. The release of platelet microparticles (PMPs) is considered of major pathophysiological significance. Objectives The aim of this study was to evaluate performances of PMP generation assay (PMPGA) compared to clinical outcome to diagnose HIT. The second objective was to compare PMPGA with performances of 14C- serotonin release assay (SRA) on the same series of patients. Methods Sera of 53 HIT-suspected patients were retrospectively incubated with citrated-whole blood from healthy donors with 1 IU and 500 IU/ml of unfractionated heparin (UH). PMPGA was performed using FACSAria® flow cytometer. The clinical diagnosis was established by two blinded independent investigators analysing in a standardized manner the patient's medical records. Performances of PMPGA and SRA (n = 53) were evaluated using ROC curve analysis with clinical outcome as reference. Results In positive HIT patients, PMPs expressing phosphatidylserine are generated with low UH concentration whereas PMP rate decreases significantly in presence of high UH concentration. Using clinical outcome as reference, sensitivity and specificity of PMPGA reached 88.9% (95% CI: 50.7-99.4) and 100.0% (95% CI: 90.0-100.0). Sensitivity and specificity of 14C-SRA were 88.9% (95% CI: 50.7-99.4) and 95.5% (95% CI: 83.3-99.2). Conclusions PMPGA is a rapid and reliable assay for HIT diagnosis. PMPGA showed good correlation with 14C-SRA performances and predominately with clinical outcome. © 2014 Elsevier Ltd. All rights reserved.


PubMed | CIMAV, University of Texas at San Antonio, Hospital Zambrano Hellion Tec Of Monterrey, Monterrey Institute of Technology and 2 more.
Type: | Journal: American journal of physiology. Heart and circulatory physiology | Year: 2017

Recent evidence has shown that nanoparticles that have been used to improve or create new functional properties for common products may pose potential risks to human health. Silicon dioxide (SiO


Poncelet P.,BioCytex | Robert S.,Aix - Marseille University | Bailly N.,Catholic University of Leuven | Garnache-Ottou F.,University of Burgundy | And 5 more authors.
Transfusion and Apheresis Science | Year: 2015

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. © 2015 Elsevier Ltd.


PubMed | Namur Research Institute for Life science NARILIS, International Consultancy in Blood Component Quality Safety Improvement, Aix - Marseille University, University of Burgundy and 2 more.
Type: Journal Article | Journal: Transfusion and apheresis science : official journal of the World Apheresis Association : official journal of the European Society for Haemapheresis | Year: 2015

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.


Douxfils J.,Namur Research Institute for LIfe science NARILIS | Chatelain B.,Namur Research Institute for LIfe science NARILIS | Hjemdahl P.,Karolinska University Hospital | Devalet B.,Namur Research Institute for LIfe science NARILIS | And 6 more authors.
Thrombosis Research | Year: 2015

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. 200 ng/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. © 2015 Elsevier Ltd. All rights reserved.


Grandjean M.,Institute Of Recherche Experimentale Et Clinique Irec | Sermeus A.,Vrije Universiteit Brussel | Branders S.,Institute of Information and Communication Technologies | Defresne F.,Institute Of Recherche Experimentale Et Clinique Irec | And 5 more authors.
PLoS ONE | Year: 2013

The expression by tumor cells of proteins with aberrant structure, expression or distribution accounts for the development of a humoral immune response. Autoantibodies (aAb) directed against tumor-associated antigens (TAA) may thus be particularly relevant for early detection of cancer. Serological proteome analysis (SERPA) aims to identify such circulating aAb through the immunoblotting of 2D-separated tumor cell proteins with cancer patient serum and the consecutive MS identification of proteins in reactive spots. This method has the advantage to use post-translationally modified proteins as a source of potential TAA. Here, we applied this strategy by using colorectal tumor cells pre-exposed to hypoxia in order to promote the expression of a pattern of TAA more likely to represent in vivo conditions. We used two human HCT116 and HT29 colorectal cancer cell lines exposed for 48 hours to 1% O2. Spots positive after immunoblotting of 2D-separated lysates of hypoxic cells with the sera of tumor-bearing mice, were collected and analysed by MS for protein identification. Among the hypoxia-specific immunogenic proteins, we identified a phosphorylated form of eukaryotic translation elongation factor 2 (phospho-Thr56 eEF2). We confirmed the increased phosphorylation of this protein in hypoxic colorectal tumor cells as well as in mouse tumors. Using a specific immunoassay, we could detect the presence of corresponding anti-phospho-Thr56 eEF2 aAb in the serum of tumor-bearing mice (vs healthy mice). We further documented that the detection of these aAb preceded the detection of a palpable tumor mass in mice and validated the presence of anti-phospho-Thr56 eEF2 aAb in the serum of patients with adenomatous polyps and colorectal carcinoma. In conclusion, this study validates a phosphorylated form of eEF2 as a new TAA and more generally, provides evidence that integrating hypoxia upstream of SERPA offers a more relevant repertoire of TAA able to unmask the presence of circulating aAb. © 2013 Grandjean et al.


Douxfils J.,University of Namur | Mullier F.,University of Namur | Mullier F.,Namur Research Institute for Life science NARILIS | Loosen C.,Namur Research Institute for Life science NARILIS | And 3 more authors.
Thrombosis Research | Year: 2012

Introduction: Rivaroxaban does not require monitoring nor frequent dose adjustment. However, searching for the optimal dose in the individual patient may be useful in some situations. Aim: To determine which coagulation assay could be used to assess the impact of rivaroxaban on haemostasis and provide guidelines for the interpretation of routine lab tests. Materials: Rivaroxaban was spiked at concentrations ranging from 11 to 1090 ng/mL in plateletpoor plasma. A large panel of coagulation assays was tested. Results: A concentration dependent prolongation of aPTT, PT, dPT, PiCT was observed. PT and dPT were the most sensitive chronometric assays but results varied depending on the reagent (Triniclot PT Excel S > Recombiplastin 2 G > Neoplastin R > Neoplastin CI + > Triniclot PT Excel > Triniclot PT HTF > Innovin). FXa chromogenic assays showed the highest sensitivity. In TGA, Cmax was the most sensitive parameter with the tissue factor induced pathway. Rivaroxaban interferes on haemostasis diagnostic tests such the measurement of clotting factors, fibrinogen, antithrombin, proteins C and S, activated protein-C resistance and Xa-based chomogenic assays. Conclusions: PT may be used as screening test to assess the risk of bleedings. A more specific and sensitive assay such as Biophen DiXaI using calibrators should be used to confirm the concentration of rivaroxaban. We also propose cut-off associated with a bleeding or thrombosis risk based on pharmacokinetic studies. Standardization of the time between the last intake of rivaroxaban and the sampling is mandatory. © 2012 Elsevier Ltd. All rights reserved.


Douxfils J.,University of Namur | Buckinx F.,University of Liège | Mullier F.,University of Namur | Mullier F.,Namur Research Institute for Life science NARILIS | And 6 more authors.
Journal of the American Heart Association | Year: 2014

Background-Signals of an increased risk of myocardial infarction (MI) have been identified with dabigatran etexilate in randomized controlled trials (RCTs). Methods and Resules-We conducted searches of the published literature and a clinical trials registry maintained by the drug manufacturer. Criteria for inclusion in our meta-analysis included all RCTs and the availability of outcome data for MI, other cardiovascular events, major bleeding, and all-cause mortality. Among the 501 unique references identified, 14 RCTs fulfilled the inclusion criteria. Stratification analyses by comparators and doses of dabigatran etexilate were conducted. Peto odds ratio (ORPETO) values using the fixed-effect model (FEM) for MI, other cardiovascular events, major bleeding, and all-cause mortality were 1.34 (95% CI 1.08 to 1.65, P=0.007), 0.93 (95%CI 0.83 to 1.06, P=0.270), 0.88 (95% CI 0.79 to 0.99, P=0.029), and 0.89 (95% CI 0.80 to 1.00, P=0.041). When compared with warfarin, ORPETO values using FEM were 1.41 (95% CI 1.11 to 1.80, P=0.005), 0.94 (95%CI 0.83 to 1.06, P=0.293), 0.85 (95% CI 0.76 to 0.96, P=0.007), and 0.90 (95% CI 0.81 to 1.01, P=0.061), respectively. In RCTs using the 150-mg BID dosage, the ORPETO values using FEM were 1.45 (95% CI 1.11 to 1.91, P=0.007), 0.95 (95% CI 0.82 to 1.09, P=0.423), 0.92 (95% CI 0.81 to 1.05, P=0.228), and 0.88 (95% CI 0.78 to 1.00, P=0.045), respectively. The results of the 110-mg BID dosage were mainly driven by the RE-LY trial. Conclusions-This meta-analysis provides evidence that dabigatran etexilate is associated with a significantly increased risk of MI. This increased risk should be considered taking into account the overall benefit in terms of major bleeding and all-cause mortality. © 2014 The Authors.


Douxfils J.,University of Namur | Chatelain C.,Namur Research Institute for Life science NARILIS | Chatelain B.,Namur Research Institute for Life science NARILIS | Dogne J.-M.,University of Namur | Mullier F.,University of Namur
Thrombosis and Haemostasis | Year: 2013

Apixaban does not require monitoring nor frequent dose adjustment. However, searching for the optimal dose for the individual patient may be useful in some situations. Moreover, there is a need for clinicians to know whether coagulation assays are influenced by apixaban use. The aim of this study was to determine which coagulation assay could be used to assess the impact of apixaban on haemostasis and provide good laboratory recommendations for the accurate interpretation of haemostasis assays. Apixaban is spiked at concentrations ranging from 5 to 500 ng/ml in platelet-poor plasma. Routinely used or more specific coagulation assays are tested. Results show a concentration dependent prolongation of aPTT, PT and dilute PT. The sensitivity mainly depends on the reagent, but none of these tests is sensitive enough to ensure an accurate estimation of the pharmacodynamic ef fect of apixaban. FXa chromogenic assays show high sensitivity and a linear correlation depending on the reagent and/or the methodology. Immunological assays and assays acting below the FXa are not influenced by apixaban. In conclusion, PT and/or dilute PT cannot be used to assess apixaban pharmacodynamic properties. More specific and sensitive assays such as chromogenic FXa assays using specific calibrators are required. In case of thrombophilia or in the exploration of a haemorrhagic event, immunological assays should be recommended, when applicable. Standardisation of the time between the last intake of apixaban and the sampling is mandatory. © Schattauer 2013.

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