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.

[Serologic diagnosis of chlamydial and Mycoplasma pneumoniae infections]. / Limites et perspectives du diagnostic sérologique à l'ère de l'amplification génique in vitro: infections génitales à Chlamydia trachomatis et infections respiratoires à Chlamydia pneumoniae et Mycoplasma pneumoniae.

The diagnosis of Chlamydia trachomatis infection can be based either on direct detection of the organism or its components or indirectly by measuring antibodies as markers of the individual's response to the infection. The latter is currently of limited value. Neither IgG or IgA antibodies can be used to diagnose current genital infection by Chlamydia trachomatis or to exclude such an infection. There is no solid ground as yet for the use of IgA antibodies as a marker of persistant or unresolved infection. Commercial tests in the Elisa format based on peptides from the MOMP of Chlamydia trachomatis are available and show good specificities and sensitivities. Hsp60 seems to have a unique role in the development of tubal scarring and antibodies to chsp60 could predict tubal factor infertility. Serology is the main diagnostic tool for the diagnosis of Mycoplasma pneumoniae infection. The serologic assays are the complement fixation test (CF), immunofluorescence, the microparticle agglutination and recently EIAs. The CF test is still used for serodiagnosis of Mycoplasma pneumoniae infection because of the sensitivity of 90%. Single titer of >or= 64 are considered to be indicative of recent infection. A number of commercial EIAs have been developped. The difficulty for IgG interpretation is a definition of a cutoff value for discriminating infected and healthy subjects. Most of the IgM assays show good diagnostic sensitivities and are valuable tools for the early diagnosis of Mycoplasma pneumoniae infection in children. There are no wholly satisfactory serological methods for diagnosis of Chlamydia pneumoniae infection. Problems arise from the high background of IgG antibody prevalence, the lack of standardized testing methods.

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%).

Neuropeptide FF in the rat adrenal gland: presence, distribution and pharmacological effects.

Neuropeptide FF (NPFF, FLFQPQRFamide) is an FMRFamide-like octapeptide exhibiting antiopiate activity. The presence of both NPFF-immunoreactivity (NPFF-IR) and NPFF-specific receptors has been described in the mammalian central nervous system (CNS). The peripheral effects of NPFF indicate that NPFF-IR material is present outside the CNS. Biochemical and immunohistochemical methods enabled us to determine the presence and distribution of NPFF-IR in the rat adrenal gland. The amount of NPFF-IR material in whole gland was estimated by radioimmunoassay to be 19.00 +/- 4.00 fmol/gland. High performance liquid chromatography analysis of adrenal extracts revealed a single molecular form which coeluted with authentic NPFF. Demedullation decreased adrenal NPFF-IR content, indicating that NPFF-IR was present in both cortex and medulla. Light microscopy revealed NPFF-IR in beaded fibers confined in the outer part of the cortex and in medullary cells. Double-labeling with antityrosine-hydroxylase and anti-NPFF antibodies showed NPFF-IR in cortical catecholaminergic postganglionic fibers restricted to the subcapsular and glomerulosa zonae. NPFF-IR was also located in medullary chromaffin cells and in rays and islets of chromaffin cells dispersed throughout the cortex. Insulin-induced hypoglycemia did not alter NPFF-IR content. Denervation lowered adrenal NPFF-IR content. These data indicate that this peptide is present in nerve fibers of extrinsic origin. In vitro approaches using adrenal slices have shown that NPFF inhibited aldosterone release in a dose-dependent manner. Taken together, these data suggest that NPFF may participate in the control of aldosterone production and adrenal blood supply.

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.

Mild hyperhomocysteinemia and hemostatic factors in patients with arterial vascular diseases.

Mild hyperhomocysteinemia, due to genetic or to environmental factors, is now recognized as a risk factor for premature arterial disease, including peripheral arterial occlusion, thrombotic stroke and myocardial infarction. It is defined by either an increased level of fasting homocysteine or by an increased level after loading with methionine, which is more frequently altered than the former. We studied the hemostatic parameters in 88 patients with premature arterial disease (mean age 43 +/- 11 years). We confirmed previously known hemostatic alterations described in vascular patients when compared to controls, but found that, among patients, some of these parameters were more altered in hyperhomocysteinemic patients. When fasting homocysteine was increased, higher alterations were found in factors VIIIc, von Willebrand and thombin-antithrombin complexes were more elevated. When post-methionine load homocysteine was increased, alterations in fibrinolytic parameters were more pronounced.

Proposal for objective evaluation of the performance of various functional APC-resistance tests in genotyped patients.

The aim of the present study was to evaluate the relative performance of five screening methods for APC resistance caused by the factor V:Q506 mutation: the original method Coatest APC Resistance Chromogenix, a modified method using the same reagents but a predilution 1+4 of the plasma in a factor V deficient plasma from Stago (Stago deficient V) or from Chromogenix (V-DEF Plasma), the Coatest APC Resistance V (Chromogenix), and Accélérimat from bioMérieux. Normalization was done against a pool of normal plasmas for the methods from Chromogenix. The study included 350 subjects, 219 were genotyped (174 FV:R506R, 42 FV:Q506R, 3 FV:Q506Q) and most of them were assessed by more than one method. Uncertainty in predicting the FV genotype was evaluated by statistical analysis, which provided a way to quantitate the performance of the different diagnostic approaches. Performance of each test was evaluated by its sensitivity, specificity, R.O.C. curves, positive and negative likelihood ratios (LR), and the overall performance was determined by two parameters derived from the LR curves : the maximum LR value obtained at the crossover of the two curves, and the distance between the two curves for LR = 10. Coatest APC Resistance V and Accélérimat were proven to be the methods most able to discriminate for factor V:Q506, while normalization was not shown to improve the screening performance. The original method from Chromogenix was confirmed to undergo many influences (factor XII, PAI-1, thrombin-antithrombin complexes, antithrombin III, hematocrit). Although a very good improvement was provided by the newest methods, they were shown to be influenced by protein S and/or factor V levels in the sample plasma.

Flow cytometry assessment of leukocyte functions in vascular pathologies.

Intravascular activation of leukocytes has been shown to be involved in a wide range of different and apparently unrelated clinical situations, such as systemic inflammatory response syndrome, ischemia/reperfusion, disseminated intravascular coagulation, atherosclerosis... All of them involve to different degrees many steps of the inflammation process, with leukocyte accumulation and release of toxic species. Haemostasis, leukocyte functions and their cross-talk are summarized in this paper, as well as the most popular methods used for studying leukocyte functions in vascular pathologies. The strengths and present limitations of flow cytometry are analyzed in comparison with the biochemical and functional approaches.

Functional upregulation of granulocytes labeled with technetium-99m-HMPAO and indium-111-oxinate.

UNLABELLED: Indium-111-oxinate-labeled granulocytes have been used in vivo for several years for the detection of abscesses. Technetium-99-m-hexamethylpropyleneamine oxime (99mTc-HMPAO) labeling has more recently been described. METHODS: The influence of radiolabeling by both radiotracers on adhesion glycoprotein CD11b quantification was studied in quiescent and formyl-methionylleucylphenylalanine (fMLP)-activated neutrophils (PMN). Adhesion was assessed on human umbilical endothelial cells (HUVEC) as well as the repercussion of the granulocyte labeling on HUVEC viability (neutral red) and metabolic activity (MTT). Chemotaxis of PMN was evaluated by measuring migration under agarose with fMLP as chemoattractant. We also measured phagocytosis and the production of hydrogen peroxide induced by staphylococcus aureus. RESULTS: Whereas whole functional integrity is maintained after labeling, most of the functions (CD11b expression, adhesion, HUVEC metabolic activity) are up-regulated while chemotaxis is decreased in the presence of both radiotracers. Indium-111-oxinate induces larger alterations than 99mTc-HMPAO. CONCLUSION: These data were obtained in normal volunteers. In patients, alterations due to the in vitro labeling procedure, in addition to potential functional alterations caused by the underlying pathology, should be taken into account during image interpretation.

Technical and biological conditions influencing the functional APC resistance test.

Poor anticoagulant response to APC is conveniently screened by a commercially available functional test (Coatest APC Resistance) allowing identification of APC-resistant patients. These patients may then be genotyped with respect to factor V, the Arg -> Gln mutation being the principle cause of APC resistance. However, determination of phenotype generally precedes that of genotype, and the need for an "abnormality threshold" prompted a study of inter-batch variations and the clinical conditions associated with an altered APC response. The response to APC was assessed twice in plasma from 111 patients using two of four successive kit batches. A modest but significant inter-batch variability was observed. At the same time, we also tested 130 patients with retinal venous occlusion (RVO), 28 patients with glaucoma and 24 normal volunteers. The APCaPTT/aPTT ratio was found to be lower in the presence of elevated thrombin-antithrombin complexes (r = 0.167, p < 0.02) and low blood viscosity (at high shear rate: r = 0.305, p < 0.0001) independently of any alteration in genotype.

Changes in phospholipid composition of blood cell membranes (erythrocyte, platelet, and polymorphonuclear) in different types of diabetes--clinical and biological correlations.

A variety of disorders of erythrocyte, platelet, and polymorphonuclear leukocyte (PMN) functions have been described in diabetes. The phospholipid composition of erythrocyte, platelet, and PMN membranes from controls and from type I and II diabetics was investigated in this study. Phospholipids were determined by densitometry using the molybdenum blue reagent. In diabetics, the relative abundance of phosphatidylethanolamine (PE) increased in all cell types studied, whereas those of sphingomyelin (Sph) and phosphatidylcholine (PC) were decreased in platelets and PMN. The percentage of phosphatidylserine (PS) was reduced in erythrocytes but increased in platelets. The level of Sph in PMN was significantly lower in type I than in type II diabetics. Moreover, the longer the duration of diabetes and the poorer the metabolic control, the greater the decrease in Sph. Rheological parameters, which reflect the behavior of red blood cells (RBC), were correlated with the alteration in PE/PS ratio in these cells.

Mechanisms underlying the cardiovascular responses to peripheral administration of NPFF in the rat.

In the present study, we examined the possibility of the presence of the Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2 (NPFF) system in the rat heart as well as the effects of drugs affecting noradrenergic transmission upon the cardiovascular responses elicited by peripheral administration of NPFF. The presence of NPFF receptors on heart sections and of NPFF-immunoreactivity in heart tissue was demonstrated with autoradiographic and radioimmunoassay procedures, respectively. Intravenous administration of NPFF (100-300 micrograms/kg) produced a dose-dependent increase in blood pressure and heart rate without affecting plasma noradrenaline and adrenaline levels. These effects of NPFF were also observed, although attenuated, in catecholamine-depleted rats and in rats pretreated with a ganglionic blocking agent, hexamethonium (10 mg/kg, i.v.). Prazosin (100 micrograms/kg, i.v.), an alpha1 adrenergic receptor antagonist, reduced the NPFF-induced blood pressure response by 50%. In contrast, propranolol (2 mg/kg, i.v.) and metroprolol (0.5 mg/kg, i.v.), beta- and beta1 adrenergic receptor antagonists, respectively, reduced the NPFF-induced heart rate response by 50%. Surprisingly, the alpha2 adrenergic receptor antagonists, idazoxan (2 mg/kg, i.v.) and yohimbine (2 mg/kg, i.v.), both produced a drastic increase in the NPFF-induced heart rate response. These data, which demonstrate the presence of the NPFF system in the rat heart, suggest that the cardiovascular responses of peripheral administration of NPFF are mediated by the stimulation of peripheral NPFF receptors. In addition, the present data show that the aforementioned NPFF-induced responses are also mediated by catecholamine-dependent mechanisms and suggest a functional interaction between adrenergic and NPFF systems.

Release of neuropeptide FF, an anti-opioid peptide, in rat spinal cord slices is voltage- and Ca(2+)-sensitive: possible involvement of P-type Ca2+ channels.

Neuropeptide FF (NPFF), an FMRFamide-like peptide with antiopioid properties, inhibits morphine-induced analgesia but also produces hyperalgesia. In the present study, the mechanisms of NPFF release were investigated in an in vitro superfusion system with rat spinal cord slices. The opening of voltage-sensitive Na+ channels with veratridine (20 microM) induced calcium-dependent NPFF release, which was abolished by tetrodotoxin (1 microM), suggesting that NPFF release depends on nerve impulse activity. We also showed that NPFF release was a function of the extent of depolarization and was calcium dependent. The 30 mM K(+)-induced release was blocked by Co2+ or Ni2+ (2.5 mM) but was unaffected by Ca2+ channel blockers of the L type--Cd2+ (100 microM), nifedipine or nimodipine (10 microM), diltiazem (20 microM), or verapamil (50 microM)--or the N type--omega-conotoxin GVIA (1 microM). In contrast, omega-agatoxin IVA (1 microM) led to a 65% reduction in NPFF release, suggesting that P-type Ca2+ channels play a prominent role. The 35% remaining release resulted from activation of an unknown subtype. The NPFF-like material in superfusates recognized spinal NPFF receptors, suggesting that NPFF release in the spinal cord has a physiological role.

Characterization of binding sites for neuropeptide FF on T lymphocytes of the Jurkat cell line.

Neuropeptide FF (NPFF) is a neuropeptide with antiopiate properties able to antagonize the action of both endogenous and exogenous opiates. Because we have recently shown that NPFF modulates the proliferation of human T lymphocytes, we have searched for binding sites for this peptide on T lymphocytes. Our study shows that T lymphocytes of the Jurkat cell line express binding sites for [125I]YLFQPQRFamide, an iodinated analogue of NPFF. This binding is time and dose dependent, reversible, saturable, and may be resolved in two distinct components of high and low affinity. The opiate receptor agonists mu, delta, and kappa, as well the antagonist naloxone, were unable to affect binding. Beside the effects of opiates on immune cells, our results suggest that an antiopiate peptide, such as NPFF, could play a role in the modulation of the immune system.