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AIMS: To assess the prognostic value of anti-apolipoprotein A-1 (anti-apoA-1) IgG after myocardial infarction (MI) and its association with major cardiovascular events (MACEs) at 12 months and to determine their association with resting heart rate (RHR), a well-established prognostic feature after MI. Anti-apoA-1 IgG have been reported in MI without autoimmune disease, but their clinical significance remains undetermined. METHODS AND RESULTS: A total of 221 consecutive patients with MI were prospectively included, and all completed a 12-month follow-up. Major cardiovascular events consisted in death, MI, stroke, or hospitalization either for an acute coronary syndrome or heart failure. Resting heart rate was obtained on Holter the day before discharge under the same medical treatment. Neonate rat ventricular cardiomyocytes (NRVC) were used in vitro to assess the direct anti-apoA-1 IgG effect on RHR. During follow-up, 13% of patients presented a MACE. Anti-apoA-1 IgG positivity was 9% and was associated with a higher RHR (P = 0.0005) and higher MACE rate (adjusted OR, 4.3; 95% CI, 1.46-12.6; P = 0.007). Survival models confirmed the significant nature of this association. Patients with MACE had higher median anti-apoA-1 IgG values at admission than patients without (P = 0.007). On NRVC, plasma from MI patients and monoclonal anti-apoA-1 IgG induced an aldosterone and dose-dependent positive chronotropic effect, abrogated by apoA-1 and therapeutic immunoglobulin (IVIG) pre-incubation. CONCLUSIONS: In MI patients, anti-apoA-1 IgG is independently associated with MACE at 1-year, interfering with a currently unknown aldosterone-dependent RHR determinant. Knowing whether anti-apoA-1 IgG assessment could be of interest to identify an MI patient subset susceptible to benefit from apoA-1/IVIG therapy remains to be demonstrated.

Adipose tissue may release mediators that induce a chronic inflammatory state and alterations in coagulation, which contribute to insulin resistance, atherosclerosis, and thrombosis. We investigated whether inflammatory and/or prothrombotic states exist in obese children and assessed their interrelationship. Sixty-one subjects were recruited, aged between 6 and 16 years, to participate in a cross-sectional study at Children's University Hospital of Geneva. Selected pro/anti-inflammatory cytokines/chemokines and hemostasis parameters were measured in obese children and lean controls. Cardiovascular risk factors in the family were indexed. Fasting glucose level, insulin, prothrombin time (PT), fibrinogen, activated partial thromboplastin time (aPTT), D-dimer, endogenous thrombin potential (ETP), C-reactive protein (CRP), interleukin-6 (IL-6), interleukin-10 (IL-10), interferon-γ-inducible-protein (IP-10), monocyte chemoattractant protein 1 (MCP-1), and interleukin-1 receptor antagonist (IL-1Ra) were measured. We estimated insulin resistance by homeostatic model assessment (HOMA). Anti- (IL-1Ra) and proinflammatory cytokines (MCP-1, IL-6) were significantly increased in obese children in comparison to the control group, even before puberty. Hemostasis was also altered in obese children with a significantly increased fibrinogen level, increased D-dimer, a shortened PT, as well as an increased ETP. No correlation was found between cytokine levels and hemostasis parameters, except for IL-6 and fibrinogen. Obese children present with inflammatory and prothrombotic states as early as 6 years of age and these states are similar in prepubertal and pubertal obese children. The cytokines IL-1Ra and MCP-1 were most significantly increased in obese children. Further investigation is necessary to determine if these cytokines, together with ETP, can reliably predict the development of diabetes and atherosclerosis.

article i nfo Introduction: Rivaroxaban (RXA) is licensed for prophylaxis of venous thromboembolism after major orthopaedic surgery of the lower limbs. Currently, no test to quantify RXA in plasma has been validated in an inter-laboratory setting. Our study had three aims: to assess i) the feasibility of RXA quantification with a commercial anti-FXa assay, ii) its accuracy and precision in an inter-laboratory setting, and iii) the influence of 10 mg of RXA on routine coagulation tests. Methods: The same chromogenic anti-FXa assay (Hyphen BioMed) was used in all participating laboratories. RXA calibrators and sets of blinded probes (aim ii.) were prepared in vitro by spiking normal plasma. The precise RXA content was assessed by high-pressure liquid chromatography-tandem mass spectrometry. For ex-vivo studies (aim iii), plasma samples from 20 healthy volunteers taken before and 2 - 3 hours after ingestion of 10 mg of RXA were analyzed by participating laboratories. Results: RXA can be assayed chromogenically. Among the participating laboratories, the mean accuracy and the mean coefficient of variation for precision of RXA quantification were 7.0% and 8.8%, respectively. Mean RXA concentration was 114±43 μg/L.RXA significantly altered prothrombin time, activated partial thromboplastin time, factor analysis for intrinsic and extrinsic factors. Determinations of thrombin time, fibrinogen, FXIII and D-Dimer levels were not affected. Conclusions: RXA plasma levels can be quantified accurately and precisely by a chromogenic anti-FXa assay on different coagulometers in different laboratories. Ingestion of 10 mg RXA results in significant alterations of both PT- and aPTT-based coagulation assays.

Summary. Background: Antiphospholipid syndrome (APS) is diagnosed by the simultaneous presence of vascular thrombosis and/or pregnancy morbidity and detection of antiphospholipid antibodies in plasma. Objectives: We have shown that prolongation of clotting time by anti-beta2-glycoprotein I (beta2GPI) antibodies correlates better with thrombosis than a positive classic lupus anticoagulant (LAC) assay in a single center study. To confirm or falsify this finding we have conducted a multicenter study. Methods and results: In 325 LAC-positive samples, we found that the beta2GPI-dependent LAC correlated 2.0 times better with thrombosis than the classic LAC assay. Although significant, this was a minimal improvement compared with the ‘classic’ LAC. It was published that calcium influences the behavior of anti-beta2GPI antibodies in coagulation assays. To investigate whether calcium plays a role in the present study, we divided the patient population into two groups: (i) blood was collected in 0.109 m sodium citrate and (ii) blood was drawn in 0.129 m sodium citrate as anticoagulant. We found that a positive result with the beta2GPI-dependent LAC assay correlated better with thrombosis [odds ratio (OR): 3.3, 95% confidence interval (CI) 1.9–5.8] when 0.109 m sodium citrate was used compared with 0.129 m sodium citrate (OR: 0.4, 95% CI 0.1–1.1). Conclusion: We were able to confirm in an international multicenter study that a positive result in a beta2GPI-dependent LAC assay correlates better with thrombosis than the classic LAC assay, but that the assay needs further study as it is sensitive to external factors such as the sodium citrate concentration used as anticoagulant in the test sample.

The presence of antiphospholipid antibodies (aPLAs) is associated with arterial or venous thrombosis and/or recurrent fetal loss. The proposed pathogenic mechanisms for aPLA effects include the inflammatory activation of monocytes and endothelial cells. Toll-like receptors (TLRs) are candidate signaling intermediates. The aim of this study was to investigate the relative contribution of TLR2 and TLR4 in cell activation by aPLAs. Of 32 patient-derived aPLAs, 19 induced an inflammatory activation of human monocytes and umbilical vein endothelial cells (HUVECs). In HUVECs, inflammatory responses to these aPLAs were increased by TNF pretreatment, which increases the expression of TLR2 but not TLR4. Anti-TLR2 but not anti-TLR4 antibodies reduced the aPLA-induced activation of monocytes and HUVECs. aPLAs activated TLR2-expressing human embryonic kidney 293 (HEK293) cells but not TLR4-expressing cells. Binding studies demonstrated an interaction between aPLAs and TLR2 but not TLR4. A role for CD14, a coreceptor for TLR2 and TLR4, can be inferred by observations that anti-CD14 antibodies reduced responses to aPLAs in monocytes, and that responses in HEK293 cells expressing TLR2 and CD14 were greater than in HEK293 cells expressing TLR2 alone. Our results demonstrate a role for TLR2 and CD14 in human endothelial cell and monocyte activation by aPLAs.

See also Mismetti P, Laporte S. New oral antithrombotics: a need for laboratory monitoring. For. This issue, pp 621–6.

One of the conclusions of the subcommittee meeting on Lupus Anticoagulant/Phospholipid dependent antibodies, held in Geneva on 2007, was the need to update the guidelines on Lupus Anticoagulant (LA) detection. Particular emphasis was given to several aspects discussed in this official communication. A new paragraph is dedicated to the patient selection, and aims to minimize inappropriate requests for LA testing. Modalities for blood collection and processing are fully delineated and the choice of tests is limited to dRVVT and a sensitive aPTT. Calculation of cut-off values for each diagnostic step are clearly stated. A final paragraph reports the interpretation of the results in general and in particular situations.

SummaryD-dimer testing is widely applied for exclusion of deep-vein thrombosis (DVT) and pulmonary embolism (PE).We report on a multicenter performance evaluation of a new particle-enhanced immuno assay Innovance™ D-Dimer. Innovance D-Dimer assay was performed in 1,543 frozen samples from outpatients suspected of DVT and/or PE enrolled in three management studies as well as in a routine clinical practice. Samples were assayed on BCS®/BCS® XP, BCT® as well as Sysmex® CA-7000, CA-1500 and CA-560 analyzers (cut-off on all analyzers: 0.5 mg/l). Stratus® CS D-Dimer and Vidas® D-Dimer Exclusion were used for comparison. The precision study indicated total coefficients of variation ranging from 2.1% to 8.4% depending on the analyzer and on the sample. Sensitivity and negative predictive values were above 99% and their lower 95% confidence interval were equal or above 97.4% and 98.6%, respectively. Specificity ranged from 38.2% to 40.4% and the respective lower 95% confidence intervals from 35.5% to 37.7%. Area under the curve was 0.90 for all assay systems except for Innovance D-Dimer with BCT (0.89).Two samples from patients with distal DVT tested negative with all assay systems. One patient with high pre-test clinical probability and proximal DVT tested negative with Vidas D-Dimer Exclusion. Our data indicate that the performances of Innovance D-Dimer, regardless of the analyzer, are similar to the reference methods, and that this assay can be used for the exclusion of venous thromboembolic disease.

Antidepressants, particularly selective serotonin reuptake inhibitors (SSRIs), are widely used for the treatment of depression and anxious disorders. The observation that depression is an independent risk factor for cardiovascular mortality and morbidity in patients with ischemic heart disease, the assessment of the central role of serotonin in pathophysiological mechanisms of depression, and reports of cases of abnormal bleeding associated with antidepressant therapy have led to investigations of the influence of antidepressants on hemostasis markers. In this review, we summarize data regarding modifications of these markers, drawn from clinical studies and case reports. We observed an association between the type of antidepressant drug and the number of abnormal bleeding case reports, with or without modifications of hemostasis markers. Drugs with the highest degree of serotonin reuptake inhibition - fluoxetine, paroxetine, and sertraline - are more frequently associated with abnormal bleeding and modifications of hemostasis markers. The most frequent hemostatic abnormalities are decreased platelet aggregability and activity, and prolongation of bleeding time. Patients with a history of coagulation disorders, especially suspected or documented thrombocytopenia or platelet disorder, should be monitored in case of prescription of any serotonin reuptake inhibitor (SRI). Platelet dysfunction, coagulation disorder, and von Willebrand disease should be sought in any case of abnormal bleeding occurring during treatment with an SRI. Also, a non-SSRI antidepressant should be favored over an SSRI or an SRI in such a context. Considering the difficulty in performing platelet aggregation tests, which are the most sensitive in SRI-associated bleeding, and the low sensitivity of hemostasis tests when performed in case of uncomplicated bleeding in the general population, establishing guidelines for the assessment of SRI-associated bleeding complications remains a challenge.

Background. Rheumatoid arthritis (RA) is a risk factor for the development of Felty's syndrome and large granular lymphocyte (LGL) leukemia. Anti-cyclic citrullinated peptide (CCP) antibodies are considered highly specific for RA and are directed against various citrullinated antigens, including citrullinated fibrinogen. Anti-CCP antibodies may interfere with the detection of citrullinated proteins and their function. In this article, we describe the possible inhibition of fibrinogen by anti-CCP antibodies with clinical consequences which have never been reported in the literature to our best knowledge. Case report. We present the case of a 79-year-old Caucasian woman with a longstanding history of untreated seropositive RA and who had been investigated for severe neutropenia since several months. The association of splenomegaly led to suspicion of Felty's syndrome. Flux cytometry was compatible with T-cell LGL leukemia. In addition, severe hypofibrinogenemia was detected. The later finding has not been consistently associated with the former clinical entities. Further investigations demonstrated that the anti-CCP antibodies of the patient also recognized the P41 peptide of citrullinated fibrinogen. The patient deceased of intracranial hemorrhage. Conclusion. It is likely, yet not definite, that high anti-citrullinated fibrinogen titers may contribute to low fibrinogen levels and could have contributed to the fatal hemorrhagic event.

Background Clopidogrel and statins are frequently coadministered in patients with ischemic heart diseases. Recent reports suggested that clopidogrel's effectiveness in inhibiting adenosine diphosphate (ADP)-induced platelets aggregation is attenuated by co-administration of certain statins. The objective of the present study was to define which statin might interfere with the antiaggregation property of clopidogrel. Methods We designed a pharmacokinetic study and tested ex vivo platelet function on 21 healthy volunteers who received clopidogrel and all currently commercially available statins: rosuvastatin [10 mg o.d.], simvastatin [20 mg o.d.], fluvastatin [80 mg o.d.], pravastatin [40 mg o.d.], and atorvastatin [20 mg o.d.]. Each statin was administered for 7 days followed by 1 week of wash-out period with clopidogrel treatment alone. Detection of the statins in the plasma was performed on all blood samples, using HPLC analytical method. Results All individuals, except one, were responders to clopidogrel with inhibition of ex vivo ADP induced platelet aggregation. All statins, except pravastatin, were detectable in the plasma at the end of each treatment period in all patients, and no statin was detectable after any of the wash-out periods. Clopidogrel was significantly less efficient to prevent platelet aggregation when coadministrated with simvastatin or fluvastatin. No difference was observed in clopidogrel efficacy when coadministered with rosuvastatin, pravastatin or atorvastatin. Conclusions This is the first study investigating clopidogrel-statin interactions on ex vivo platelet function with all commercially available statins and which were administered to the same individuals. It demonstrates in healthy volunteers that at the doses used in this study, simvastatin and fluvastatin, but not atorvastatin, pravastatin or rosuvastatin interfere with the anti-aggregation effect of clopidogrel.

Summary. Background: D-dimer (DD) measurement has proved to be very useful to exclude venous thromboembolism (VTE) in outpatients. However, during pregnancy, the progressive increase as well as the interindividual variations of DD means that in this instance they are of poor value to rule out VTE. Only a few studies have reported measurements of DD levels in the postpartum. Objectives: To measure DD sequentially in the puerperium in order to determine when DD levels return to values obtained in non-pregnant women and can again be used in the exclusion of VTE. Patients and methods: After uncomplicated pregnancies, 150 women delivering at term either vaginally (n = 100) or by cesarean section (n = 50) were included. DD levels were measured immediately following delivery and next at days 1, 3, 10, 30 and 45. Results: There was a marked elevation of DD at delivery, especially when instrumental. All DD measurements were above 500 ng mL−1 at delivery, at day 1 and at day 3 postpartum. A sharp decrease in DD was observed between day 1 and day 3, followed by a slight increase at day 10. At day 30 and day 45, respectively, 79% and 93% of women in the vaginal delivery group and 70% and 83% in the cesarean group had levels below 500 ng mL−1. Bleeding, breastfeeding and heparin prophylaxis did not modify DD levels significantly. Conclusion: Using the Vidas DD new assay, our study provides reference intervals for DD in the postpartum period. Using a cut-off at 500 ng mL−1, DD measurement for ruling out VTE was found to be useful again 4 weeks after delivery.

SummaryThe aim of this study was to evaluate the agreement in assay results between commercial kits for the measurement of anti- β2glycoprotein I antibodies. Ten manufacturers provided one IgG and one IgM kit to three testing centres. Samples from patients with primary (n=13) or secondary (n=3) antiphospholipid syndrome (APS), from lupus patients without APS features (n=6) and from normal individuals (n=2) were tested in the three centres according to manufacturers’ instructions. Dilutions in normal serum of a pool made from positive patients’ samples (Forum Calibrators) and dilutions of humanized monoclonal antibodies (MoAbs) were used as additional calibrators. The calibration curves obtained with each calibrator differed widely between kits. The rate of positivity of patients’ samples varied from 7 to 16 for IgG and from 2 to 17 for IgM, depending on the kit. Perfect agreement occurred in 12/22 samples for IgG and 5/22 samples for IgM. Samples from normals were found negative by all kits. Between kits, cutoff values varied up to five fold when expressed in Forum Calibrators arbitrary units and up to three fold when expressed in MoAbs equivalents. Examination of discrepant samples indicated that about half of the discrepancies, scoring 8:2 and 9:1, involved the same few kits. In highly discrepant samples, some kits appeared as high responders as compared to others. In conclusion, with the exception of a few kits, agreement in assay results was acceptable. In conclusion, additional efforts are however necessary, especially concerning the way to assess the cutoff point and the adoption of a reference calibrator, in order to improve standardization of the assays.

The biological criteria of the antiphospholipid syndrome defined at the Sapporo meeting in 1998 included the presence of lupus anticoagulant (LA) and/or anticardiolipin antibodies at medium and high titers. During the 48th SSC meeting held in Boston July 2002, it was proposed to modify these criteria. Four patient groups were defined, the first one comprising LA and anti-beta2glycoprotein I antibodies (abeta2GPI), the second one LA only, the third one abeta2GPI only and the fourth one other antiphospholipid antibodies such as antiprothrombin, anticardiolipin, antiphosphatidylethanolamine, etc. This proposition raised the issue of the association of abeta2GPI with APS clinical criteria (thrombosis and pregnancy morbidity). In some studies, a strong association between IgG abeta2GPI and thrombosis was found, whereas in others this association could not be demonstrated. In the obstetrical field, few studies are available and no clear conclusion can be drawn yet. However, for thrombosis or pregnancy morbidity, it has been shown that in up to 10% of patients, abeta2GPI are the sole antibodies present and therefore the diagnosis of APS would be missed in these patients. In addition, some studies suggest that the severity of disease is dependent on the number of positive tests and on their titers. We recommend abeta2GPI assays to be included in the panel of antiphospholipid screening tests. However, the standardisation of abeta2GPI assays has to be improved in order to ensure better comparability between the studies.

Summary. Background: Inhibitors of HMG-CoA reductase are widely used to prevent atherosclerosis progression. The expression of adhesion molecules on activated endothelial cells (EC) is an important step in the initiation and progression of atherosclerosis. Objectives: We investigated whether adhesion molecule expression on activated EC is influenced by simvastatin, fluvastatin and pravastatin and, if so, by which mechanisms. Methods: Human EC from umbilical veins or saphenous veins were pretreated overnight with statins with or without mevalonate, and also for simvastatin or fluvastatin with the isoprenoid intermediates, farnesyl pyrophosphate (FPP), or geranylgeranyl pyrophosphate (GGPP). After 4–6 h activation with tumor necrosis factor (TNF)-α or lipopolysaccharide (LPS), surface adhesion molecule expression was evaluated by ELISA and by flow cytometry. The same experiments were performed with selective inhibitors of geranylgeranyltransferase (GGTI-286) and farnesyltransferase (FTI-277). Results: Pretreatment with simvastatin, fluvastatin or pravastatin potentiated the TNF-α and LPS-induced expression of E-selectin and VCAM-1, and mevalonate reversed the potentiating effect of these statins. GGPP also reversed the potentiating effect of simvastatin or fluvastatin on adhesion molecule expression, while FPP only partially reversed this effect. Furthermore, GGTI-286, but not FTI-277, mimicked the effect of simvastatin by increasing the TNF-α-mediated overexpression of E-selectin. Conclusions: Statins increase E-selectin- and VCAM-1-induced expression on vascular endothelial cells stimulated with TNF-α or LPS. The inhibition of geranylgeranylated proteins could contribute to this effect.

Cases of passive alloimmune thrombocytopenia have been reported due to transfusions of blood products with antiplatelet antibodies. The aim of our studywas to search for antiplatelet antibodies in HPA-homozygous blood donor women once pregnant and also to perform, in case of positivity, a retrospective analysis of platelet counts of the recipients of their blood products. HPA-1, -2, -3 and -5 genotyping were performed on 500 platelet donors (42% women). Circulating antiplatelet antibodies were screened for byMAIPA assayin 122 women who experienced at least one pregnancyand who were homozygous for either HPA-1a, -1b, -3a, -3b, -5a or -5b. None of the women homozygous for HPA-1 or -3 had circulating antiplatelet antibodies. In contrast, two of the 98 women homozygous for HPA-5a and one of the two women homozygous for HPA-5b had circulating antibodies. A retrospective analysis of the medical charts of the 37 recipients of 55 blood components from these three women showed no case of passive alloimmune thrombocytopenia. Our study indicates the presence of plateletspecific antibodies in 2.5% of HPA-homozygous female platelet donors who were previously pregnant. Although none of the recipients developed passive alloimmune thrombocytopenia, this aspect of blood transfusion safetyshould be addressed bya large prospective trial. The Hematology Journal (2003) 4, 441–444. doi:10.1038/sj.thj.6200338

To compare and evaluate the potential benefit of two different anticoagulation regimens during endotoxemia in an ovine model.Animal prospective randomized and controlled study following preliminary dose-range study conforming with the Guide for the Care and Use of Laboratory Animals as promulgated by the Council of the American Physiologic Society and reviewed by the Ethical Committee for Animal Research of our institution.Laboratories of anesthesiological investigations and hemostasis, primary care university medical center.Twenty-two adult sheep of either sex, weighing 29-42 kg (mean 35.8 kg), surgically instrumented for chronic studies and randomly allocated to receive three different treatment groups: unfractionated heparin (40 units.kg.hr; n = 7), recombinant hirudin (500 units.kg.hr; n = 7), or saline (nonanticoagulated controls; n = 8).Ovine model of severe endotoxin shock induced by a continuous intravenous endotoxin infusion over 72 hrs (10 ng.kg.min ).Endotoxin infusion alone produced a progressive hypotensive, hyperdynamic shock state with right ventricle failure leading to the death of all animals of the control group within 44 hrs (median, 12 hrs). Heparin profoundly improved survival rate in this model, whereas effective anticoagulation with hirudin did not significantly increase the survival rate compared with controls. Animals in the control group died from disseminated intravascular coagulation, metabolic acidosis, and hemorrhagic pulmonary edema, whereas sheep treated with hirudin died from severe pulmonary edema (hypoxemia, increased alveoloarterial oxygen gradient associated with an increased wet/dry lung weight ratio) in the absence of disseminated intravascular coagulation.We conclude that systemic anticoagulation with the thrombin inhibitor hirudin is without benefit in this sheep model of lethal endotoxemia, whereas anticoagulation with heparin affords not only effective prevention of endotoxin-induced coagulation disorders but also global protection with prevention of respiratory decompensation and thus markedly improves survival rate in this situation.

Resume La coagulation peut etre divisee schematiquement en trois phases, l’initiation, l’amplification, enfin la formation d’une quantite importante de thrombine. De nombreux activateurs sont impliques dans chacune de ces etapes. Pour que la balance hemostatique soit maintenue, des inhibiteurs existent, le premier (inhibiteur de la voie du facteur tissulaire) bloquant l’initiation de la coagulation, le second (systeme de la proteine C) l’amplification et le troisieme (antithrombine) bloquant principalement la thrombine. En cas de coagulopathie intravasculaire disseminee (CIVD), de nombreux changements sont observes, tant au niveau des activateurs que des inhibiteurs. Cet article revoit la coagulation en phase d’equilibre puis en cas de derangement extreme (CIVD) et decrit, en prenant comme exemple le sepsis severe, les nombreux liens existant entre la coagulation et l’inflammation.

SummaryInter-laboratory variability of anti-β2-glycoprotein I antibody measurements (IgG and IgM) was investigated in the frame of the European Forum on Antiphospholipid Antibodies and its Standardization Group. Twenty-eight samples from patients with autoimmune diseases, two samples from blood donors and a set of six calibrators obtained by dilution with normal plasma of a pool of patient samples were sent to 21 European centers. Six of them used commercial kits and the others home-made assays. Marked differences in the steepness of the calibration curves obtained with the calibrator provided were observed. The standard deviations of sample measurement were high. Cut-off of positivity varied from 7 to 90 Forum Units (FU) for IgG and from 10 to 138 FU for IgM, whereas the rate of positivity varied from 50 to 93% for IgG and from 13 to 70% for IgM. No clear relationship between cut-off values and positivity rate could be established for either isotype. Adopting a common cut-off did not markedly improve the overall agreement between centers in positive/negative sample classification. Because of the majority of low positive samples, excellent concordance between centers (as defined by kappa values from 0.8 and 1) occurred only in 13% of cases for IgG and in 6% of cases for IgM, because many selected samples were low-positive. Despite the large variability of anti-β2-glycoprotein I measurements between centers, the agreement on results with highand medium-positive samples was good.

SummaryAntiphospholipid antibodies (aPLA) are associated with thrombophilia and recurrent pregnancy loss. They bind directly to anionic phospholipids or via phospholipid-binding proteins such as β2-glycoprotein 1 (β2GP1). The underlying mechanisms by which aPLA induce a thrombophilic phenotype are not well understood.The present work was done to determine whether antibodies to β2GP1 activate endothelial cells (EC) and whether NFκB is involved in this activation. Incubation of EC with these antibodies resulted in a redistribution of NFκB from the cytoplasm to the nucleus after a delay of several hours. This was accompanied by an increased expression of tissue factor and of the leukocyte adhesion molecules ICAM-1, VCAM-1 and E-selectin. Inhibition of the nuclear translocation of NFκB abolished the response to these antibodies. In comparison to anti-β2GP1 antibodies, incubation of EC with TNF resulted in a more rapid (within 30 minutes) redistribution of NFκB and a more pronounced expression of tissue factor and of the leukocyte adhesion molecules. The slower response to the antibodies as compared to TNF suggests that the NFκB response to anti-β2GP1 antibodies is indirect.Taken together our results imply that NFκB is an essential intermediate in the activation of EC by anti-β2GP1 antibodies.

Congenital afibrinogenemia is a rare autosomal recessive coagulation disorder characterized essentially by bleeding symptoms, but miscarriages and, paradoxically, thromboembolic events can also occur. Most reported mutations leading to congenital afibrinogenemia are located in FGA encoding the fibrinogen A α-chain. In this study, we analysed 12 individuals from a consanguineous Syrian family with reduced or absent fibrinogen levels: those with fibrinogen levels around 1 g/l (n = 7) were found to be heterozygous for a novel frameshift mutation in FGA exon 5 (c.1846 del A) and those with undetectable fibrinogen levels (n = 5) were homozygous for the same mutation. This novel frameshift mutation is the most C-terminal causative FGA mutation identified to date in afibrinogenemic patients. The resulting aberrant Aα-chain (p.Thr616HisfsX32) is most likely synthesized, but is less efficiently assembled and/or secreted into the circulation given the phenotype of asymptomatic hypofibrinogenemia in heterozygous individuals and bleeding diathesis in homozygous individuals.

SummaryAntiphospholipid antibodies (APLA) are associated with thrombophilia and recurrent pregnancy loss. Different mechanisms have been proposed to explain their pathogenic effects and among them, we have previously shown that APLA accumulate in late endosomes of human umbilical vein endothelial cells (HUVEC) leading to a redistribution of the cation-independent mannose-6-phosphate receptor (CI-M6PR). Because many APLA are directed towards β2-glycoprotein 1 (β2GP1)-phospholipid complexes, we investigated the localisation of β2GP1 in HUVEC. By immunofluorescence analysis, using monoclonal and polyclonal anti- β2GP1 antibodies, we detected β2GP1 at the cell surface and in late endosomes. Incubation of HUVEC with anti- β2GP1 antibodies resulted in antibody accumulation at the cell surface and within late endosomes and in a redistribution of the CI-M6PR from the Golgi apparatus to late endosomes. The anti- β2GP1 antibodies remained detectable in late endosomes even after several days of incubation in antibody-free medium. The accumulation of anti- β2GP1 antibodies in late endosomes of endothelial cells and the resulting modification of intracellular protein trafficking may contribute to the pathogenic effects of these antibodies.

SummaryQuantification of lupus anticoagulant (LA) in clinical samples is hampered by the lack of a suitable standard of activity. We evaluated the use of mAbs displaying LA activity for this purpose. As most patient samples contain both β2Glycoprotein I (β2GP1) and prothrombin dependent LA, a combination of two mAbs, one of each specificity, was added to normal plasma in a concentration from 0 to 60 g/ml. Eight assay systems using different reagents and instruments were used. The calibration curves were linear for all but one, with marked differences between the responsiveness to each mAb. A panel of plasmas from 69 patients with persistent LA diagnosed using the SSCISTH criteria was tested. An antiphospholipid syndrome (APS) was present in 40, whereas 29 were asymptomatic. LA activities of individual plasmas varied between assays (p

We determined the prevalence of factor V Leiden and of prothrombin G20210A mutations in a cohort of unselected outpatients (n = 748) referred for suspected deep vein thrombosis (DVT) and/or pulmonary embolism (PE) and a pooled analysis of similar studies was also performed. Based on the clinical presentation, the prevalence of factor V Leiden was 15·7% in the 83 patients with DVT and 14·1% in the 99 patients with PE compared with 5·3% in patients without DVT and/or PE (control group). The prevalence of the prothrombin G20210A mutation did not differ among the three groups (3·9% for controls, 4·8% for DVT and 3·9% for PE patients). We then divided the 99 patients with PE by separately analysing those with PE but without DVT (n = 57) and those with PE and DVT (n = 42). Compared with the control group, the prevalence of factor V Leiden was 10·5%, odds ratio (OR) 2·10 [95% confidence interval (95% CI) 0·68–5·45] in patients with primary PE and 19·1%, OR 4·20 (95% CI 1·54–10·30) in patients with DVT and PE. For the prothrombin G20210A mutation, no statistically significant differences were found between the control group and the three other groups. In conclusion, our data and the pooled analysis indicate that patients with primary PE are less often affected by the factor V Leiden mutation. No statistically significant differences were observed between patients and controls for the prothrombin G20210A mutation.

Resume Malgre les efforts deployes depuis son apparition, la standardisation du test de dosage des anticorps dits anticardiolipine n'a pas pu etre encore realisee. En consequence, les resultats obtenus avec les trousses commerciales sont difficilement comparables et les coefficients de variation importants. Des progres recents ont permis de mieux caracteriser l'heterogeneite de ces anticorps et de differencier leurs cibles antigeniques. L'utilisation d'antigenes et de composants purifies va permettre d'ameliorer la situation actuelle.

SummaryLess than 60 cases of acquired factor (F)XIII deficiencies have been reported, most having distinct clinical features. To illustrate the therapeutic challenges of acquired FXIII inhibitors, we report a case of a 65-year-old patient with no previous bleeding history who suddenly developed massive haemorrhages associated to a strong and isolated FXIII inhibitor. No underlying disorder has been detected till now after three years of follow-up. Despite aggressive treatment with prednisone, rituximab, cyclophosphamide, immunoglobulin, immunoadsorption and immune tolerance his inhibitor is still present, although at low titre and with a clinical benefit since the patient has no more bleed since more than one year. Moreover the patient had a venous thromboembolic complication. After a review of the management of acquired FXIII deficiency patients and based on the management of acquired haemophilia we discuss a possible strategy for such difficult cases.

Background While the assessment of analytical precision within medical laboratories has received much attention in scientific enquiry, the degree of as well as the sources causing variation between them remains incompletely understood. In this study, we quantified the variance components when performing coagulation tests with identical analytical platforms in different laboratories and computed intraclass correlations coefficients (ICC) for each coagulation test. Methods Data from eight laboratories measuring fibrinogen twice in twenty healthy subjects with one out of 3 different platforms and single measurements of prothrombin time (PT), and coagulation factors II, V, VII, VIII, IX, X, XI and XIII were analysed. By platform, the variance components of (i) the subjects, (ii) the laboratory and the technician and (iii) the total variance were obtained for fibrinogen as well as (i) and (iii) for the remaining factors using ANOVA. Results The variability for fibrinogen measurements within a laboratory ranged from 0.02 to 0.04, the variability between laboratories ranged from 0.006 to 0.097. The ICC for fibrinogen ranged from 0.37 to 0.66 and from 0.19 to 0.80 for PT between the platforms. For the remaining factors the ICC’s ranged from 0.04 (FII) to 0.93 (FVIII). Conclusions Variance components that could be attributed to technicians or laboratory procedures were substantial, led to disappointingly low intraclass correlation coefficients for several factors and were pronounced for some of the platforms. Our findings call for sustained efforts to raise the level of standardization of structures and procedures involved in the quantification of coagulation factors.

The performance of a new membrane ELISA for semi-quantitative determination of plasma D-dimer has been evaluated. Its cut-off is about 500 ng/ml FEU and this single test is completed within 10 min. D-dimer was measured in 301 patients suspected of pulmonary embolism by conventional microplate and membrane ELISA. For the latter, readings were made by eye and some differences were noticed between the readers for reactions in the grey zone. Sensitivity and negative predictive values were similar for the two ELISA (higher than 90%). The 95% confidence intervals of sensitivity and negative predictive values obtained with this membrane ELISA suggest that this new test may be used as a diagnostic tool to exclude the presence of pulmonary embolism.

SummarySome lupus anticoagulants (LA) have been shown to be directed against phospholipid-bound prothrombin. While developing an ELISA to detect anti-prothrombin autoantibodies in patient serum or plasma, no or very low signal was observed using human prothrombin immobilized on plain polystyrene plates. In contrast, the same LA-positive samples bound specifically to prothrombin coated on γ-irradiated plates, depending on the radiation dose, in the absence of added calcium and phospholipid. Optimization of the assay required the addition of 0.1% Tween 20 to the buffers. Antibody specificity for immobilized prothrombin was ascertained by competition using liposome-bound prothrombin, since fluid-phase prothrombin competed poorly. Seventy-seven of 139 patients (55.4%) with LA related to a variety of underlying diseases possessed anti-prothrombin antibodies (27 IgG, 35 IgM and 15 both isotypes), either isolated or more often associated with anti-(β2 glycoprotein I (β2GPI) antibodies. These included 67-71% of the patients with systemic lupus erythematosus and related disorders, primary antiphospholipid antibody syndrome or drug-induced LA (autoimmune groups), but only 19-20% of those with infection or malignancy (p

SummaryThe performances of nine commercial kits and an in-house method (HM) for the quantitation of anticardiolipin antibodies (ACA) have been evaluated in a multicenter study. Ninety control and patient samples and six standards from Louisville University were run with kits and with the HM. Marked differences in positivity rate between kits were observed, ranging from 31 to 60% for IgG and 6 to 50% for IgM. Concordance between kits occurred in 59 and 51% of samples for IgG and IgM respectively. Concordance coefficients (kappa) ranged from 0.13 to 0.92. Slopes of regression lines between the declared units of Louisville standards and the units measured from the calibrators of the kits showed great diversity and ranged from 0.159 to 0.931 for IgG and from 0.236 to 0.836 for IgM. The β2-glycoprotein I (β2-GPI) content of the dilution buffers and the wells supplied with the kits revealed noticeable differences. However samples containing anti-(β2-GPI antibodies were classified similarly by all but one kit. In contrast the ability to measure samples devoid of anti-β2-GPI antibodies differed markedly between the kits.This study shows that differences in positivity rates between the commercial kits may contribute to the differences in ACA prevalence rate found in the literature. The choice of cut-off levels may partly explain the moderate concordance between the kits. In addition some samples behave very differently depending on the kits. In spite of the expression of results in PL units, standardization of ACA assays has not been achieved.

The aim of this study was to compare four house standards coming from two University Hospital laboratories with the standards provided by the Antiphospholipid Standardization Laboratory (ASL) in order to quantify anticardiolipin antibodies. Using two different plates and two different buffered protein solutions, slopes from the serial dilutions of each of the four house standards were found comparable. In contrast different slopes were obtained when using the ASL standards which consist of a mixture of sera. Our results indicate that dilutions of single sera are more suitable than mixture of sera when quantification of anticardiolipin antibodies is required.

Plasma D-Dimer (DD), a highly sensitive marker of venous thromboembolism, was measured with an ELISA assay preoperatively and on the 12th postoperative day in 173 patients undergoing major hip surgery (78 elective arthroplasties and 95 operations for fractures). Proximal deep venous thrombosis (DVT) was detected by systematic compression venous ultrasonography on the 12th postoperative day in 12 (7%) patients. In one additional case, proximal DVT was diagnosed by venography. Preoperative DD level was significantly higher in patients with fracture than in patients undergoing elective arthroplasty. At a cutoff of 500 micrograms/L as determined by ROC curve analysis, the sensitivity, specificity, positive and negative predictive values of the pre-operative DD concentration for the development of subsequent proximal DVT were 93%, 23%, 36% and 96%, respectively. The diagnostic exclusion value of the DD measurement on the 12th postoperative day was similar but for a cutoff of 2000 micrograms/L. These data suggest that plasma DD measurement might be useful to predict and diagnose proximal DVT following major hip surgery.

We have investigated whether the levels of thrombin-activatable fibrinolysis inhibitor (TAFI) were correlated with D-dimer levels during pregnancy and at delivery. From the 10th week of pregnancy to delivery, 519 samples from 144 women (mean age 29.3 +/- 5, range 19-43) were obtained. We confirm the gradual increase of D-dimer levels, and provide reference intervals for D-dimer measurements throughout normal pregnancy. TAFI levels increased moderately during pregnancy but no inverse correlation with D-dimer levels was observed.

SUMMARY BACKGROUND: Anticardiolipin (aCL) and anti-beta(2)glycoprotein I (abeta(2)GPI) antibodies are part of the criteria for antiphospholipid syndrome (APS). Therefore they are widely measured and about 30 commercial kits are available. OBJECTIVES: To investigate the analytical and clinical performances of four fully automated, chemiluminescent assays: HemosIL AcuStar aCL IgG, HemosIL AcuStar aCL IgM, HemosIL AcuStar abeta(2)GPI IgG, and HemosIL AcuStar abeta(2)GPI IgM. METHODS: Cut-off values were assessed by testing 250 blood donors. Total coefficients of variation (CV) were determined with six plasma pools and two controls ranging from 4.3 to 2694 U mL(-1) depending on the assay. Samples from 218 well-characterized patients and 103 controls were measured in three laboratories to determine inter-laboratory variation. The results of the 321 samples were compared with three commercial assays (REAADS, INOVA and VARELISA). RESULTS: Cut-off values were assigned to 20 arbitrary units for all the tests. Total CV ranged from 4.3 to 11.2%. No interference of hemoglobin, bilirubin, triglycerides, heparins and rheumatoid factor was observed. Inter-laboratory variability was low and no sample changed status. Overall status agreement between HemosIL assays and the comparator kits ranged from 82 to 96%. Sensitivity, specificity, agreement when predicting APS and the odds ratios when predicting a thrombotic or obstetric event gave comparable results between HemosIL AcuStar and the three other assays. CONCLUSIONS: Our study demonstrates that the fully automated HemosIL AcuStar aPL assay panel showed similar performances to the three commercial ELISAs commonly used by various laboratories to detect antiphospholipid antibodies.

Clopidogrel has a known biological variability that has been consistently associated with recurrence of coronary ischemic events in clinical studies. Among the tests that are currently available, quantification of the phosphorylation status of the vasodilator phosphoprotein (VASP assay) is probably the most specific assay to evaluate the inhibition of the P2Y12 receptor by clopidogrel. A genetic polymorphism of the cytochrome 2C19 has been associated with the biological efficacy of clopidogrel and is also associated with recurrent ischemic events. The VASP assay and the 2C19 genotyping are candidates for the identification of patients at risk; this is the focus of the present review.

The effects on hemostasis of two high-flux membranes in hollow-fiber configuration, polyamide (PAM) and polyacrylonitrile (AN69), were analyzed in a cross-over study involving ten chronic hemodialyzed patients. Blood samples were obtained at arterial and venous sites of the extracorporeal circuit before dialysis and at 15, 30 and 180 min. Primary hemostasis: PAM induced an early significant drop in platelet counts, but at 180 min there was no longer any difference between membranes. Beta-thromboglobulin release by PAM was significantly higher at all time points. Coagulation: thrombin-antihrombin III complexes (TAT) and fibrinopeptide A increased significantly, the highest values being found with AN69. With both membranes the arteriovenous differences in TAT levels were negative throughout the sessions. Fibrinolysis: no significant differences were observed. In conclusion, both membranes induced hemostatic changes. Although these two hollow-fiber dialyzers look relatively similar, the changes observed were different, polyamide acting mainly on primary hemostasis and polyacrylonitrile on coagulation.

SummaryBecause the use of radioactive fibrinogen uptake test (FUT) has become questionable both for ethical (risk of virus transmission) and technical (lack of sensitivity) reasons, we investigated the potential value of two alternative methods for screening of asymptomatic deep venous thrombosis following elective digestive surgery: liquid crystal contact thermography (LCCT) and measurement of plasma concentration of D-dimer (DD), as compared with bilateral ascending phlebography. Out of 194 patients, 185 underwent phlebography on the 8th (0-19, median and range) postoperative day. Despite prophylaxis with low-molecular-weight heparin and elastic stockings, DVT was detected on phlebography in 58 legs of 45 patients. Sensitivity of LCCT with respect to the presence of DVT was 55% (n = 184 patients) or 28% (n = 368 legs) with a specificity of 67% and 82%, respectively. These poor performances were obtained despite a good interobserver agreement for the LCCT assessments (overall kappa coefficient of 0.66 between three experts). The most accurate cut-off of DD for discriminating patients with or without DVT was 3,000 pg/1, as determined by ROC curve analysis. Sensitivity of a DD level of more than 3,000 pg/1 for the presence of phlebographically documented DVT on the 8th postoperative day was 89% for a specificity of 48%.Thus, LCCT cannot be used for screening of postoperative, mainly asymptomatic DVT following general surgery. On the other hand, measurement of plasma DD may be useful for initial screening, a negative result (level less than 3,000 pg/1) allowing to exclude DVT (negative predictive value of 93%) and a positive result (positive predictive value of 35%) requiring confirmation by phlebography. This sequential approach might be useful in studies of the efficacy of antithrombotic regimens for prophylaxis of DVT in patients at risk.

Anticardiolipin (aCL) and anti-beta2 glycoprotein I (anti-beta2GPI) assays are widely performed because they are part of the laboratory criteria for the antiphospholipid syndrome (APS). Despite several standardization workshops and the availability of a worldwide accepted calibrator material for aCL, a high variability in numerical assay results and in sample classification is still observed in comparative studies and external quality control surveys. For anti-beta2GPI assays, comparison of numerical values is impeded by the absence of a common calibrator material, and external quality surveys similarly show a large overlap in sample classification. Numerous variables impact assay results, among them the source and integrity of beta2GPI, the secondary calibration process, and the assessment and derivation of cutoff values. For both assays, the vast majority of laboratories use commercial kits whose number has risen considerably in the past years. However, many problems persist, and there is a need to improve the comparability in assay results. The use of monoclonal antibodies as reference calibrators has to be especially considered, with their suitability evaluated by future collaborative studies and external quality controls surveys. Manufacturers should provide more precise information on results obtained when testing control groups for establishing reference ranges and cutoff values. From the customers' perspective, it is important that each laboratory, even if using commercial kits, assesses its local cutoff value whenever possible. In the field of autoimmunity, assay standardization is a difficult but nevertheless important task. Much more effort is needed to reduce the high interlaboratory variability in assay results even if absolute standardization cannot be feasibly achieved.

Human endothelial cells (EC) express Toll-like receptor 4 (TLR4), a receptor for lipopolysaccharides (LPS), but little or no TLR2, a lipopeptide receptor. The aim of this study was to investigate to what extent inflammatory stimuli modify the expression by EC of TLR4 and TLR2, of the TLR2 co-receptors TLR1 and TLR6 and of the TLR2-accessory proteins CD14 and CD36. Stimulation of umbilical vein derived EC with TNF-alpha, LPS or IL-1beta for 24h induced a strong increase in TLR2 mRNA but not in TLR1, TLR4 and TLR6 mRNA. Inflammatory activation had little effect on CD14 mRNA, but decreased the expression of CD36 mRNA. TLR2 antigen was readily detected by flow cytometry on activated EC, but not on resting EC. A significant proportion of TLR2 was found to be located intracellularly. By using specific signalling pathway inhibitors we established that the induction of TLR2 by inflammatory stimuli was dependent on NF-kappaB, p38-MAP kinase and c-Jun kinase. IRAK-1 phosphorylation after treatment with 10mug/ml of lipoteichoic acid (LTA), a TLR2 agonist, was only observed in TNF-alpha-stimulated EC and not in resting EC. Furthermore, LTA potentiated the increase of the inflammatory markers E-Selectin or IL-8 in EC pre-treated with TNF-alpha, LPS or IL-1beta, but not in resting EC. These results imply that the up-regulated TLR2 is functionally active. Interestingly, LTA had no effect on TLR2 expression, nor maintained TLR2 expression, in activated EC. This suggests that lipopeptide responses of EC are dependent on the continued presence of inflammatory cytokines, provided by other cell types, or LPS. In conclusion, inflammatory stimuli induce a high TLR2 expression in EC, which in turn enables the cells to strongly respond to lipopeptides. The up-regulation of TLR2 may be of relevance for the vascular effects of Gram-positive bacteria.