Multiplate® evaluation of acetylsalicylic acid efficacy in carotid surgery: routine and genetic influencing factors.

Essentials Acetylsalicylic acid (ASA) is prescribed to patients scheduled for carotid endarterectomy (CEA). We measured ASA efficacy during CEA by Multiplate® and searched for influencing factors. Most patients scheduled for CEA and treated by ASA are sensitive to this therapy. Influencing genomic factors are involved in ASA metabolism and in platelet function modulations. SUMMARY: Background Acetylsalicylic acid (ASA) is recommended before, during and after carotid endarterectomy (CEA). The efficacy of ASA is influenced by numerous biological and genotypic factors. Objectives To determine the biological efficacy of ASA by using the Multiplate® method, and to explore the biological parameters and genomic factors influencing this efficacy. Methods This descriptive cross-sectional study included all patients scheduled for CEA between January 2012 and April 2013. Multiplate® tests were performed at day 0 and day 30. A set of 66 single-nucleotide polymorphisms (SNPs) from 38 genes or DNA regions were selected and studied along with phenotypic parameters by the use of hierarchical clustering (HC) for multidimensional data management. Results Fifty-five patients receiving ASA were analyzed. Of the patients, 95% were found to be sensitive to ASA, with values under the threshold of normality (400 AU min-1 ). However, there were notable differences in residual aggregation among subjects over a wide range. HC revealed four subclusters comprising three categories of parameters: (i) routine and functional parameters - in ASA-treated patients, the ASPItest was highly linked to the ADPtest, to platelet count, and, to a lesser extent, to fibrinogen and hematocrit; (ii) polymorphisms in genes involved in ASA absorption and in the arachidonic acid pathway (ABCB1 and COX-1); and (iii) polymorphisms in genes modulating basal platelet function, i.e. TBXA2R, ADRA2A, PEAR1, ITGA2 and ITGB1. Conclusion Most patients treated with ASA before CEA were sensitive to it, according to Multiplate® ASPItest results. Genomic factors influencing this efficacy are SNPs involved in ASA absorption and metabolic pathway, and in modulations in basal platelet function.

Rotation thromboelastometry detects thrombocytopenia and hypofibrinogenaemia during orthotopic liver transplantation.

BACKGROUND: Orthotopic liver transplantation can be associated with haemorrhage, particularly in patients with severe liver dysfunction. We assessed the value of rotation thromboelastometry (ROTEM) to monitor coagulation in the operating theatre, its correlation with routine laboratory findings, and its ability to guide platelet (Plt) and fibrinogen (Fg) transfusion. METHODS: Twenty-three patients were included in this prospective observational study. Laboratory tests and ROTEM tests (EXTEM, INTEM, FIBTEM, and APTEM) were performed six times during the procedure. Correlations between laboratory findings and ROTEM parameters were sought. Thresholds for ROTEM parameters were determined with receiver-operating characteristic (ROC) curve analysis according to Plt count and Fg levels. RESULTS: Clot amplitude at 10 min (A10) of EXTEM was well correlated with Plt count and Fg levels (R(2)=0.46 and 0.52, respectively, P<0.0001). FIBTEM A10 was correlated with Fg (R(2)=0.55, P<0.0001). ROC analysis showed that EXTEM A10 with a threshold of 29 mm predicted thrombocytopenia with a sensitivity of 79% and a specificity of 60%, and a threshold of 26 mm predicted hypofibrinogenaemia with a sensitivity of 83% and a specificity of 75%. CONCLUSIONS: ROTEM is useful for the global assessment of coagulation in the operating theatre. EXTEM was the most informative for assessing the whole coagulation process and A10 showed value in guiding Plt and Fg transfusion.

A role for thrombin in liver fibrosis.

BACKGROUND/AIM: Several lines of evidence incriminate the serine proteinase thrombin in liver fibrogenesis either through its procoagulant function or its signaling via cell-surface receptors. The aim of this study was therefore to evaluate the effect of thrombin inhibition on experimental liver fibrosis. METHODS: Fibrosis was induced in rats by administration of CCl4 for either three or seven weeks. Oral administration of the thrombin antagonist SSR182289 started one week after the start of CCl4 intoxication. Fibrosis and the area occupied by alpha smooth muscle actin (ASMA) positive cells were quantified with histomorphometry. Expression of fibrosis related genes was measured by real time RT-PCR. RESULTS: After three weeks of CCl4, treatment with SSR182289 did not significantly decrease the area of fibrosis but significantly decreased the area of ASMA positive cells by 22% (p = 0.03) and the expression of TIMP-1 mRNA by 52% (p = 0.02). There was no effect on gene expression of collagen I, MMP-2, or TIMP-2. After seven weeks of CCl4, treatment with SSR182289 resulted in a significant decrease in fibrosis (-30%, p = 0.04) and ASMA positive areas (-35%, p = 0.05). SSR182289 alone had no effect on the measured parameters. Additionally, it did not alleviate the acute toxicity of CCl4 as shown by measuring levels of serum aminotransferases and the area of necrosis. CONCLUSIONS: These data provide evidence that thrombin antagonism can reduce liver fibrogenesis. The early effect of SSR182289 on ASMA and TIMP-1 expression suggests that it is beneficial in reducing fibrogenic cell activation.

[Imaging of the lower limb veins: when and how?]. / Quand et comment réaliser une imagerie des veines des membres?

Evaluation for possible lower limb deep venous thrombosis (DVT) is a very frequently requested examination. In France, imaging diagnosis is essentially based on complete Doppler sonographic evaluation of both lower limbs. In patients with no co-morbid condition, the D-dimer assay is useful to exclude the possibility of DVT. A positive diagnosis of DVT is based on the lack of venous compressibility and abnormal Doppler signal. The diagnostic accuracy relies on adequate knowledge of vascular anatomy and sufficient training, especially at the calf level. For experienced sonographers, the accuracy is similar at the thigh and calf level. In patients with suspected pulmonary embolus, evaluation of the lower extremity veins is mandatory and frequently performed with CT immediately following CT pulmonary angiography. However, this examination has not been validated yet.

Assessment of the 20210 G to A prothrombin variant in a sample of patients from the French Basque Country with various thrombophilic conditions.

We investigated the distribution of the prothrombin variant G20210A (PT20210A) in a sample of 103 patients (mean age: 34.5 years) living in the French Basque Country and presenting with conditions known to be significantly associated with this peculiar mutation according to a literature review. These patients suffered from repeated personal or familial venous thromboses, stroke at young age, or repetitive maternal-fetal disorders (abortions, preeclampsia, fetal growth retardation). Five patients (4.8%) were found to be carriers: two homozygotes and three heterozygotes (one also heterozygote for factor V Leiden). Of note, two presented with mesenteric venous thrombosis. The distribution of PT20210A among our sample was comparable to data from Western European series albeit a tendency for lower mutation prevalence was observed in our subgroup with obstetrical disorders. In addition, no significant difference in PT20210A frequency was evidenced between autochthonous Basques and individuals from other origins.

Changes in haemostasis after laparoscopic surgery in gynaecology: contribution of the thrombin generation test.

Surgery induces immediate hypercoagulability by direct alteration of the vascular bed, release of procoagulant substances from the extravascular spaces and blood flow decrease, and delayed hypercoagulation in response to tissue damage which triggers inflammatory responses. Thus, the postoperative period represents a high-risk time for thrombosis. Recognition of high-risk individuals would make it possible to improve thromboembolism prevention. We studied in women undergoing laparoscopic surgery a series of markers known to be related to the thrombotic risk and confronted their results with those of a global test, the thrombin generation test (TGT) described by Hemker's group. Our results show that two groups of patients can be distinguished according to usual risk markers (PAI-1, TAT, body mass index): the higher risk group demonstrates higher initial TGT values, but also a postoperative decrease of the TGT values whose mechanisms remain to be defined.

Tissue factor mRNA quantitation without prior monocyte isolation.

Monocyte tissue factor (TF) quantitation evaluates the involvement of coagulation processes in many diseases. However, technical difficulties, such as blood sampling of cells representative of the whole intravascular pool, cell isolation, protein quantitation or activity assessment, hinder reliable evaluation of TF expression by activated monocytes. Early determination of such activation can be achieved through TF mRNA quantitation by RT-PCR and sensitive product detection, such as automated electrophoresis of fluorescently labeled products. Although it is very sensitive, this method has its limitations. It needs to be standardized using other mRNA that display two main characteristics: the absence of upregulation during inflammation and similar levels of expression when compared with the target mRNA. Widely used standardization housekeeping genes such as HLA or GAPDH genes only meet the former requirement. We demonstrate here that CD11b gene expression meets both conditions. Moreover, because of its specific expression in myelomonocytic cells, it is possible to avoid further monocyte purification from a regular mononuclear cell preparation. A rapid, sensitive, specific and accurate way to evaluate monocyte TF expression is described in this paper.

Rapid ELISA D-dimer testing in the exclusion of venous thromboembolism in hospitalized patients.

The clinical diagnosis of deep-vein thrombosis (DVT) and pulmonary embolism (PE) is known to be unreliable. Until now, no biological marker has been found to confirm thrombosis, but help can be gained from a biological marker ruling out the diagnosis of DVT or PE, i.e., the sensitive measurement of D-dimer (DD) species. This article summarizes our experience in introducing a rapid D-dimer test (ELISA VIDAS D-dimères test, bioMérieux) in a collaborative strategy for thrombosis diagnosis during 9 consecutive months involving 1,131 measurements. The efficacy of the DD test was very different according the type of patient, and departments where the DD test provides a real diagnostic benefit were identified. High clinical probability for thrombosis was encountered in 32 patients and radiology was carried out, although D-dimer was negative: none of these patients was found to have a thrombosis after radiologic examination. However, extensive progress must be made in test prescription to reduce the excessive rate of positive D-dimer tests (78%) and positive measurements that are not followed up by radiology (42%).

Strategies for the safe and effective exclusion and diagnosis of deep vein thrombosis by the sequential use of clinical score, D-dimer testing, and compression ultrasonography.

Patients with suspected deep vein thrombosis (DVT) are subjected to leg vein compression ultrasonography (CUS) that confirms DVT in only 20 to 30% of patients. A positive CUS is consistent with DVT irrespective of clinical score. The sequential use of a simple clinical score assessment, a rapid sensitive enzyme-linked immunosorbent assay (ELISA) D-dimer test and CUS to safely exclude DVT is promising. The clinical score is a validated clinical model of complaints, signs, and symptoms, on the basis of which a pretest clinical probability for DVT can be estimated as low, moderate, and high. The safe exclusion of DVT by a rapid sensitive D-dimer test in combination with clinical score or CUS necessitates a negative predictive value of more than 99%. The negative predictive value for DVT is determined by the sensitivity of the rapid ELISA D-dimer test and the prevalence of DVT in subgroups of outpatients with suspected DVT. The prevalence of DVT in outpatients with a low, moderate, and high clinical score varies widely from 3 to 10%, 15 to 30% and more than 70%, respectively. A negative rapid ELISA D-dimer and a low clinical score (prevalence DVT 3 to 5%) will have a very high negative predictive value of more than 99.5% to exclude DVT without the need of CUS testing. A negative ELISA D-dimer test and a first-negative CUS safely exclude DVT in patients with a moderate clinical score with a negative predictive value of more than 99.5%, therefore obviating the need to repeat CUS. The use of a rapid ELISA D-dimer testing in patients with a high clinical score is not recommended. A negative CUS, a low clinical score, and a positive ELISA D-dimer, even less than 1000 ng/mL exclude DVT with a nega tive predictive value of more than 99%. Patients with a negative CUS, but a positive ELISA D-dimer, and a moderate or high clinical score have a probability of DVT of 3 to 5% and 20 to 30%, respectively, and are thus candidates for repeated CUS testing. The proposed sequential use of the clinical score assessment, a rapid ELISA D-dimer test, and CUS will be the most cost-effective diagnostic strategy for DVT because of a significant reduction of CUS examinations and gain of time for the patient and physician in charge.

Quantification of the inflammatory response in exudates to three polymers implanted in vivo.

Flow cytometry was used to quantify an inflammatory reaction in vivo as a new approach to evaluating the biocompatibility of biomaterials. The exudate formed inside cylindrical tubes composed of polyvinyl chloride (PVC), silicone elastomer (SIL), or polyurethane (PU) implanted subcutaneously in the dorsal region of rats was collected over a 3-week period. The volume, number of cells, and concentration of fibrinogen were determined in the exudate for the three biomaterials. The exudate was analyzed using a flow cytometry technique after labeling of the leukocytes with a monoclonal anti-CD45 antibody. Fibrinogen rose progressively over the 3-week period for the three polymers. After the different leukocyte lines were identified in rat blood samples, their determination in the exudate revealed differences among the three biomaterials. At day 2, PVC induced a predominantly neutrophilic inflammatory reaction whereas PU and SIL gave a mixture of monocytes and neutrophils. At day 9, the aspect of the cytograms was different, but the identification of the subpopulations was still possible. At day 23, the number of cell events became too low to distinguish the subpopulations. An even more detailed approach might be possible using specific labeling for each leukocyte line to establish a comparison among the three biomaterials. Flow cytometry associated with histomorphometric assessment might provide a precise quantitative in vivo test for determining the biocompatibility of materials.

D-dimer strategy in thrombosis exclusion--a gold standard study in 100 patients suspected of deep venous thrombosis or pulmonary embolism: 8 DD methods compared.

DD are now recognized as a valuable tool to screen patients suspected of deep venous thrombosis or pulmonary embolism before carrying out a gold standard radiologic examination. The newest methods available claim to be able to ascertain the absence of thrombosis, but they have yet to prove their efficiency. We compared the performances of 3 reference ELISA methods (D-DI Asserachrom Stago, D-dimer Enzygnost Behring and Dimertest GOLD EIA Agen), 5 recent rapid methods (VIDAS D-Dimer bioMérieux, Instant IA Stago, Simplired Agen, Nycocard D-dimer Nycomed and Accuclot D-Dimer Sigma Diagnostics) and two routine latex methods (Dimertest American Diagnostica and FDP-Slidex bioMérieux) in 100 patients. One of the rapid quantitative methods was demonstrated to have a level of efficiency comparable to that of ELISA methods. Finally, the cost and efficiency of different strategies were evaluated, the association of a routine latex method with the VIDAS D-Dimer bioMérieux being proven to be the most efficient.