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Introduction VTE‐BLEED is a validated score for identification of patients at increased risk of major bleeding during extended anticoagulation for venous thromboembolism (VTE). It is unknown whether VTE‐BLEED high‐risk patients also have an increased risk for recurrent VTE, which would limit the potential usefulness of the score. Methods This was a post hoc analysis of the randomized, double‐blind, placebo‐controlled PADIS‐PE trial that randomized patients with a first unprovoked pulmonary embolism (PE) initially treated during 6 months to receive an additional 18‐month of warfarin vs. placebo. The primary outcome of this analysis was recurrent VTE during 2‐year follow‐up after anticoagulant discontinuation, that is, after the initial 6‐month treatment in the placebo arm and after 24 months of anticoagulation in the active treatment arm. This rate, adjusted for study treatment allocation, was compared between patients in the high‐ vs. low‐risk VTE‐BLEED group. Results In complete case analysis (n = 308; 82.4% of total population), 89 (28.9%) patients were classified as high risk; 44 VTE events occurred after anticoagulant discontinuation during 668 patient‐years. The cumulative incidence of recurrent VTE was 16.4% (95% confidence interval [CI], 10.0%‐26.1%; 14 events) and 14.6% (95% CI, 10.4%‐20.3%; 30 events) in the high‐risk and low‐risk VTE‐BLEED groups, respectively, for an adjusted hazard ratio of 1.16 (95% CI, 0.62‐2.19). Conclusion In this study, patients with unprovoked PE classified at high risk of major bleeding by VTE‐BLEED did not have a higher incidence of recurrent VTE after cessation of anticoagulant therapy, supporting the potential yield of the score for making management decisions on the optimal duration of anticoagulant therapy.

SummaryPortal vein thromboses are frequent in cirrhotic patients and may be favoured by hypercoagulability in the splanchnic venous system. The coagulation balance and thrombin generation (TG) were evaluated in platelet-free plasma obtained from portal and systemic blood samples in 28 cirrhotic patients while undergoing transjugular intrahepatic porto-systemic shunt. TG assay (TGA) was performed with all samples from cirrhotic patients and with plasma samples from 14 healthy controls, with varying concentrations of tissue factor and phospholipids, with or without thrombomodulin. Screening tests and specific assays were also performed and activated partial thromboplastin time was shorter in portal plasma samples with higher FVIII and lower protein C levels, well correlated with Child-Pugh scores, and higher D-dimers and F1+2 levels However, all TGA parameters were similar in portal and jugular samples, possibly due in part to similar concentrations of factor II and antithrombin in these two sites of plasma sampling. TGA showed lower thrombin peaks and endogenous thrombin potential values in cirrhotic plasma compared to those of healthy controls. Importantly, a resistance to thrombomodulin that well correlated with factor VIII and PC levels, was evidenced in all samples from patients with cirrhosis, and was more significant in those from severely affected cases. This study therefore supports the existence of a relative hypercoagulability in the portal vein of cirrhotic patients that is likely due to protein C/S deficiency and to high FVIII levels.

BACKGROUND: After stopping a 3 to 6 months course of oral anticoagulation for a first episode of idiopathic venous thromboembolism (VTE), the risk of recurrent VTE is high (10% per year). In this setting, international guidelines recommend at least 6 months treatment. However, this recommendation is not satisfactory for the following reasons: (1) no randomized trial has compared 6 months to extended duration (2 years) anticoagulation; and (2), even though the frequency of recurrent VTE is similar after pulmonary embolism (PE) and deep vein thrombosis (DVT), the fatality rate of recurrent VTE after PE is higher than that after DVT. METHODS: A French multicentre double blind randomized trial. The main objective is to demonstrate, after a first episode of symptomatic idiopathic PE treated for 6 months using a vitamin K antagonist, that extended anticoagulation for 18 months (INR between 2 and 3) is associated with an increased benefit / risk ratio (recurrent VTE and severe anticoagulant-related bleeding) compared to placebo. The double blind evaluation is ensured using by active warfarin and placebo, and blinded INR. The protocol was approved by the ethics board of the Brest Hospital on the 7th of March 2006. For an alpha risk of 5% and a beta risk of 20%, the estimated sample size is 374 patients. EXPECTED RESULTS: This study has the potential to: (1) demonstrate that the benefit / risk ratio of extended anticoagulation for 18 months is higher than that observed with placebo in patients with a first episode of idiopathic PE initially treated for 6 months, during and after the treatment period; and (2) to validate or invalidate the contribution of isotope lung scans, lower limb Doppler ultrasound and D-Dimer at 6 months of treatment as predictors of recurrent VTE (medico-economic analysis included).

SummaryRetinal vein occlusion (RVO) is a multifactorial disease involving vessel damage, stasis, viscosity and thrombosis. Conflicting findings on hereditary thrombophilic risk factors have been reported and their impact on RVO features remains to be defined. The aim of the present study was to evaluate the prevalence of hereditary thrombophilic risk factors (HTRF) and characteristics of RVO in patients with or without HTRF. The design of the study was a prospective, observational case series. Two hundred and thirty-four patients with RVO were included consecutively. A French healthy population of the same region was studied as control group. The HTRF studied were protein C (PC), protein S (PS) and antithrombin (AT) deficiencies, factor V Leiden (FVL) and factor II 20210A polymorphisms. Chi-Square was used for comparison with the healthy subjects and between RVO patient with and without HTRF according to localisation (branch vs. central), type of RVO (ischemic or non-ischemic), recurrence, age at first event and classical vascular risk factors. Twenty-two patients had HTRF (12 FV Leiden heterozygotes, 9 FII 20210A heterozygotes and 1 PS deficiency). No AT or PC deficiency was detected. Frequencies of PS deficiency, FVL and FII 20210A allele were similar to the reference population as well as to published data in the general caucasian population. Eighty-six patients experienced their first episode before the age of 60 years. Systemic hypertension, glaucoma and angina were significantly less frequent in patients with RVO before 60 years. Fourteen of the 22 patients with one HTRF (64%) experienced their first episode of RVO before the age of 60 years compared to 72 of 212 without HTRF (34%) (p = 0.006). Heterozygote status for FV Leiden was significantly more frequent in patients who had experienced their first episode of RVO before 60 years (p = 0.027). In conclusion, this study suggests a role of FV Leiden in the occurrence of RVO in patients younger than 60 years who exhibit fewer acquired vascular risk factors than in older patients.

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

Introduction Bien que les examens d’hemostase usuels refletent mal le risque hemorragique des patients cirrhotiques operes, des transfusions de PFC sont parfois systematiques pour en corriger les anomalies. Cette pratique est controversee en raison de l’augmentation de la volemie induite aggravant l’hypertension portale et le risque hemorragique. L’utilisation de CCP apportant les facteurs de coagulation sous faible volume est proposee. Aucune etude n’a cependant evalue son efficacite chez les patients au cours d’une THO. Le TGT refletant plus globalement l’equilibre hemostatique que les tests d’hemostase usuels, nous avons analyse les parametres du TGT apres addition in vitro de PFC ou de CCP aux echantillons de plasmas de patients cirrhotiques preleves au cours d’une THO. Patients et methodes Cette etude a ete menee sur fonds de tube dans le cadre du soin courant. Dix patients, 7H/3F, âge median 62,5 ans, Child median 11 [8–13], taux de prothrombine (TP) median 36 % [32–62] ont ete preleves a differents temps peroperatoire : avant incision (P1), en fin d’hepatectomie (P2), avant declampage porte (P3) et 30 minutes apres reperfusion (P4). Les TGT (methode CAT, concentrations finales de facteur tissulaire et de phospholipides respectivement 5 pM et 4 μM, PPP reagent® Stago) ont ete realises sur les plasmas deplaquettes avant et apres ajout de PFC (5 mL/kg) ou de CCP (Confidex® 12,5 UI/kg ou 25 UI/kg). Les resultats ont ete compares a ceux d’une population de sujets sains (n = 29 ; 22 H/7 F) (test de Mann et Whitney) et pour chaque prelevement avant et apres surcharge des plasmas (test de Wilcoxon). Resultats Les parametres du TGT des patients avant incision (P1) ainsi qu’a P2 et P3 sont similaires a ceux des sujets sains : ETPP1 basal min–max [939–1584] nM*min vs ETPsujets sains [1040–1914] nM*min p = 0,24. En revanche a P4 une generation de thrombine significativement plus basse est objectivee : ETPP4 basal [678–1222] nM*mn p Nous avons evalue l’effet de ces produits sur le TGT au temps P1 en raison de l’injection de PFC souvent realisee des le debut de la procedure. Le PFC n’entraine pas d’effet significatif ETPP1 + PFC [1008–1760] nM*mn p = 0,10 vs P1 alors que l’addition de CCP induit un effet significatif : ETPP1 + CCP 12,5 UI/kg [1920–3083] nM*mn p = 0,002 vs P1 avec une hypercoagulabilite des la plus faible concentration. Discussion Le TGT permet d’objectiver un etat hemostatique equilibre chez les patients cirrhotiques que ne reflete pas le TP. L’inefficacite du PFC a corriger la generation de thrombine in vitro a P4 est un argument qui soutient la non prescription peroperatoire systematique de ce produit sanguin. A l’inverse, l’efficacite du CCP in vitro plaide en faveur de son utilisation lors de la reperfusion (P4) mais non en debut de procedure (P1) en raison d’un risque thrombotique comme le suggere la generation tres majoree de thrombine.

SummaryThe recommended therapeutic range of International Normalized Ratio (INR) for oral anticoagulant treatment in patients with the antiphospholipid syndrome remains controversial. As a part of this controversy, it has been suggested that lupus anticoagulants (LA) could interfere with the determination of prothrombin time, thus questioning the validity of monitoring the treatment of these patients using INR. To clarify this point, we compared the values of INR obtained in the plasmas of two groups of patients, one without LA (n = 47), and the other with LA (n = 43). INR were determined using 8 different thromboplastin reagents on the same automated coagulation instrument. Chromogenic factor X, which is supposed to be insensitive to the presence of LA, was also measured. The results are the following: provided INR was calculated using calibrated reference plasmas, there was no significant difference between INR values obtained with the 8 reagents, both in the non-LA and in the LA groups (CV: 5.9 and 6.7%, respectively). Closer examination revealed that INR results obtained with one reagent (the recombinant thromboplastin Innovin) diverged from those of the 7 others, leading to an overestimation of INR, to a very large extent in some instances. However this effect was restricted to a subset of the patient population with LA (6 out of 43). Finally, the relationship between INR (average value obtained using the 8 reagents) and factor X was identical in non-LA and in LA patient groups. We conclude that, provided the reagents which display the LA interference are identified and excluded for this purpose, the INR system is valid for monitoring oral anticoagulant treatment in patients with LA.

Heparin-induced thrombocytopenia (HIT) is frequently associated with antibodies (Abs) to heparin-PF4 complexes (H-PF4). In order to investigate whether there are variations in specificity of Abs, we studied 63 samples from patients with suspected HIT. Two groups of samples were separated after comparing their reactivity against H-PF4 or recombinant PF4 (r-PF4) using ELISA. In group Ab1 (n = 46), Abs only or mainly bound to H-PF4 complexes and thus most of the epitopes recognized probably involved both heparin and PF4. In group Ab2 (n = 17), Abs exhibited similar reactivity to r-PF4 and H-PF4, and the antigens recognized were possibly neoepitopes mainly expressed by modified PF4 and by H-PF4 complexes. Platelet activation tests were positive with 56 samples containing high titres of Abs to H-PF4. Most samples (n = 59) contained IgG antibodies, often associated with IgA antibodies which were more frequently found in group Ab2, and/or IgM. With unfractionated heparin treatment, HIT was associated with Ab1 or Ab2 antibodies, whereas only Ab1 antibodies were detected after low-molecular-weight heparin (LMWH). Furthermore, cross-reactivity with danaparoid sodium was present only in group Ab1 and mainly involved LMWH-treated patients.

Blood coagulation proteins were determined in 285 healthy fetuses from 19 to 38 weeks' gestation and compared with those of 60 normal full- term newborns and 40 adult controls. Prolongation of the coagulation screening tests, prothrombin time, activated partial prothrombin time, and thrombin clotting time, in fetuses throughout intrauterine life was explained by low levels of vitamin K-dependent factors (II, VII, IX, and X), contact factors (XI, XII, prekallikrein, and high-molecular- weight kininogen), factor V, factor VIII, and fibrinogen. Low levels of antithrombin III, heparin cofactor II, protein C and protein S, and tissue factor pathway inhibitor were also found, and these probably contributed to a satisfactory hemostatic balance. Some of these parameters were evaluated by both immunologic and functional assays to detect possible “fetal” proteins. An increase in factor levels was observed after the thirty-fourth week of intrauterine life for most of the coagulation activators and inhibitors, but only factors V and VIII reached adult values at birth. This study therefore showed that fetal hemostasis is a dynamic system that evolves gradually toward the neonatal state and then toward the adult state.

Resume L'exploration de l'hemostase chez un patient avec antecedents hemorragiques est indispensable. Un examen clinique et un interrogatoire precis sont essentiels et permettent de guider le diagnostic vers une pathologie de l'hemostase acquise ou constitutionnelle en precisant les circonstances de survenue et les caracteristiques du saignement ainsi que l'existence d'antecedents familiaux. Ils doivent etre systematiquement associes a la pratique de test biologiques d'orientation comprenant un hemogramme et une numeration plaquettaire, un temps de saignement (TS), un temps de cephaline plus activateur (TCA), un temps de Quick (TQ ou TP), un dosage du fibrinogene et un temps de thrombine (TT). L'analyse combinee des resultats de tous ces tests guide le biologiste vers des dosages specifiques necessaires a l'identification precise d'une anomalie. La decouverte d'une pathologie constitutionnelle de l'hemostase, notamment d'une maladie de Willebrand ou d'une hemophilie, impose d'adresser le patient a un centre specialise. Une information sur la maladie, son traitement et les conduites a tenir doit etre faite au patient et a sa famille par une equipe de specialistes qui etablira une carte personnalisee pour chaque patient et les membres de sa famille. Une enquete genetique et un suivi regulier de ces patients doivent imperativement etre effectues. L'absence d'anomalies du bilan d'orientation chez un patient presentant une histoire personnelle et/ou familiale de saignements bien documentee doit entrainer des investigations supplementaires. Les caracteristiques du saignement guideront vers une maladie de Willebrand dont certaines formes sont particulierement difficiles a diagnostiquer ou une coagulopathie moderee ou encore vers une anomalie de la fibrinolyse ou du facteur XIII. En l'absence de diagnostic positif, une pathologie vasculaire devra etre evoquee.

International audience; OBJECTIVE: To assess the rate of early (first trimester) and late (second and third trimester) fetal loss in women who are factor V Leiden homozygous. METHODS: Between December 1995 and February 2007, consecutive, unrelated white women who were factor V Leiden homozygous and who had been pregnant at least once were recruited from 10 French hemostasis units. For reasons of comparison, we included women who were factor V Leiden heterozygous and a group of noncarriers. The frequency of early and late fetal loss was assessed retrospectively and compared among the three groups. The effect of concomitant thrombophilic abnormalities was evaluated. The overall pregnancy outcome was reported. RESULTS: We analyzed 240 thromboprophylaxis-free pregnancies in 95 women who were factor V Leiden homozygous, 425 in 195 women who were factor V Leiden heterozygous, and 182 in 73 women who were noncarriers. The risk of late fetal loss was higher in women who were homozygous (13/95, 13.7%) compared with those who were noncarriers (1/73, 1.4%, odds ratio 11.41, 95% confidence interval 1.46-89.46, P=.002), whereas it was similar in women who were heterozygous and in noncarriers (6/195, 3.1% compared with 1/73, 1.4%, P=.68). The percentage of women with early fetal loss was similar in the three groups (P=.81). The live-birth rate was 80%, 84%, and 85%, respectively, for women who where homozygous, heterozygous, and noncarriers (P=.88). CONCLUSION: The factor V Leiden homozygous genotype increases the risk of late fetal loss. However, the overall likelihood of a positive outcome is high in our series of women who were homozygous. LEVEL OF EVIDENCE: III.

Heparin-associated thrombocytopenia and thrombosis is a severe complication of systemic heparin therapy. Its treatment is mainly based upon discontinuation of heparin therapy. However in some patients requiring emergency cardiac or vascular surgery, reexposure to heparin may be unavoidable. We report the management of two such patients by use of antiplatelet drugs for a vascular procedure. In the two cases, a combination of iloprost, a stable prostacyclin analogue (1 to 2 ng/kg/mn) with aspirin and dipyridamole was shown to inhibit ex vivo the heparin-induced platelet aggregation. These antiplatelet agents were continued during the perioperative period. A successful vascular procedure was achieved with full heparinization without subsequent thrombocytopenia or thrombotic or hemorrhagic complications. This experience supports the hypothesis that heparin can be readministered early to patients with heparin-associated thrombocytopenia and thrombosis, provided antiplatelet therapy is given.

A nucleotide 807T variant of the glycoprotein Ia gene that correlates with increased platelet surface levels of the platelet collagen receptor alpha2beta1 was recently found to be associated with an increased risk of ischaemic stroke in younger patients. We report the history of twins who developed ischaemic strokes and were shown to be homozygous for the alpha2 807T allele. The twins developed ischaemic strokes at the ages of 23 and 33 years, one of them with recurrent events. They had no conventional risk factors. Cardiac and vascular investigations were normal and no aetiology could be found. There was a family history of cerebrovascular disease. Genotyping of glycoprotein alpha2 C807 T was performed and both twins were found to be homozygous for the 807T allele. This allele probably contributed to the occurrence of strokes in these young men. Further prospective studies are needed to evaluate whether screening for this polymorphism should be considered in young patients with unexplained stroke, particularly when a positive family history was found.

Assay of factor VIII (FVIII) in patient samples is routinely carried out using the one-stage assay rather than the chromogenic substrate assay. The introduction of new FVIII preparations for the treatment of haemophilia A, including immunopurified FVIII and particularly, recombinant FVIII (rFVIII) concentrates, has led to discrepancies between the results obtained with the two assays. In patients treated with rFVIII concentrates, FVIII levels measured with the one-stage assay can be 20-50% lower than those measured with the chromogenic assay. In this study, the one-stage assay was performed with cephalin dilutions higher than those recommended by the manufacturer. B-domain-deleted recombinant FVIII, Refacto, was diluted to eight different concentrations, ranging from 1-100 IU dL(-1), in FVIII-deficient plasma and the FVIII activity of the eight solutions was determined by the chromogenic method in a central laboratory. Aliquots were then assayed by the one-stage method in the four participating laboratories, using different dilutions of CK-Prest. When CK-Prest was reconstituted according to the manufacturer's recommendations (dilution 1 : 1), the difference between the one-stage and chromogenic methods was close to 30%. CK-Prest cephalin dilutions of 1 : 5 and 1 : 8 gave very similar results with the two methods, without increasing the interlaboratory coefficient of variation. These findings confirm the influence of phospholipids on the one-stage assay, particularly the importance of using a phospholipid concentration close to the physiological value in platelets. This modified one-stage method may therefore offer an alternative to the use of a concentrate-specific standard.

The common genetic G to A variation of the prothrombin gene is associated with elevated levels of prothrombin [factor II (FII)] and is recognized as a risk factor for thrombosis. To determine whether one type of assay for plasma FII measurement was more efficient than other assays in displaying high FII levels in 20210A carriers, we compared five methods of measuring FII levels [i.e. an enzyme-linked immunosorbent assay (ELISA), a standard clotting assay, and three chromogenic methods using three different activators: Ecarin, Oxyuranus, and Textarin] in 30 G20210A patients and 30 G20210G controls. Plasma concentrations of factor X and factor VII + factor X were also determined by a clotting procedure. Functional assays were found to be equally efficient in demonstrating significantly higher FII levels in 20210A carriers than in non-carriers (P < 0.0001). With ELISA, the difference observed was less significant (P < 0.005). The specificity of every assay increased with FII cut-off levels; when a cut-off of 115% was applied, sensitivities of functional assays were between 73 and 93%, while sensitivities of ELISA declined dramatically to 33%. FII/factor X and FII/factor VII + factor X ratios were significantly higher in 20210A carriers (P < 0.0001). In conclusion, functional assays are preferentially required for measurements of FII levels in carriers of the 20210A variant.

Ventilation/perfusion pulmonary scintigraphy (PS), the current mainspring in the diagnosis of pulmonary oedema (PE), is frequently non-conclusive. The objective of this study was to determine, prospectively, the diagnostic value of the association of D-dimers and helicoidal thoracic scanner (HTS) in a continuous series of ambulatory adults with suspected PE and admitted to a cardiologic emergency unit.Exclusion criteria were as follows: symptoms or clinical signs of seventy, contraindication for HTS and/or on pulmonary angiography, curative anticoagulant therapy for more than 48 hours, history of PE less than 3 months before or the impossibility of conducting all the examinations with 48 hours. All the patients underwent determination of D-dimers by rapid ELISA test, HTS and 2 reference examinations, venous Doppler of the lower limbs and a PS, completed by pulmonary angiography if the PS did not permit diagnosis and the venous Doppler was negative.One hundred and six patients were selected over a recruitment period of 18 months. The prevalence of PE was of 45% (48/106). Forty-four of the 48 PE of the series were central form. HTS was considered dubious in 10 patients (10.4%, PE+ n = 2, PE- n = 8). The results of D-dimers were negative in only 34.5% patients without PE (20/58). Pulmonary angiography was necessary in 15 patients. The negative and positive predictive values of D-dimers were respectively of 100 and 55.8% (48/86) and those of HTS respectively 100 (46/46) and 92% (46/50).The diagnostic strategy of clinical suspicion of PE, starting with determination of D-dimers by rapid ELISA test followed by helicoidal thoracic scanner in the case of a positive result, was particularly effective in this series of patients with a high prevalence of PE. These results must be confirmed in a larger series and in a general emergency unit.

Quantification 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 beta2Glycoprotein I (beta2GP1) 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 microg/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 SSC-ISTH criteria was tested. An antiphospholipid syndrome (APS) was present in 40, whereas 29 were asymptomatic. LA activities of individual plasmas varied between assays (p10(-4)), but homogeneous subgroups were identified. In a majority of samples, LA activity displayed a prothrombin-dependent profile, with a variable contribution of beta2GP1-dependent activity. The latter was associated to beta2GP1 antibodies detected by solid-phase immunoassay. By using 3 dilute Russell viper venom time assays, higher LA titers were found in APS, compared to asymptomatic patients (p0.05).

The value of the platelet aggregation test, carbon 14-labeled serotonin release assay (SRA), and heparin-platelet factor 4 enzyme-linked immunosorbent assay (H-PF4 ELISA) for the diagnosis of heparin-induced thrombocytopenia was evaluated by studying blood samples from 100 patients with suspected heparin-induced thrombocytopenia, and categorized into 4 clinical groups: unlikely (n = 22), possible (34), probable (36), and definite (8) thrombocytopenia. Results of the platelet aggregation test were positive in 40 of 44 patients with probable or definite heparin-induced thrombocytopenia (sensitivity 91%) and in 5 of 22 unlikely to have heparin-induced thrombocytopenia (specificity 77%). The SRA exhibited sensitivity of 88% and negative predictive value of 81%, close to those values for the platelet aggregation test; specificity and positive predictive value were 100%. The sensitivity of the heparin-PF4 ELISA was 97%, with specificity 86%, and a positive correlation was recorded between the level of antibodies to H-PF4 and clinical score (P = 0.66). When ELISA was used with the platelet aggregation test or SRA, positive predictive value and specificity were 100% when both tests yielded positive results, and negative predictive value was 100% when both tests yielded negative results. A biologic flow chart was designed that presented a choice based on the results of the platelet aggregation test or SRA in association with ELISA, and enabled more accurate and specific identification of heparin-induced thrombocytopenia.

The recommended therapeutic range of International Normalized Ratio (INR) for oral anticoagulant treatment in patients with the antiphospholipid syndrome remains controversial. As a part of this controversy, it has been suggested that lupus anticoagulants (LA) could interfere with the determination of prothrombin time, thus questioning the validity of monitoring the treatment of these patients using INR. To clarify this point, we compared the values of INR obtained in the plasmas of two groups of patients, one without LA (n = 47), and the other with LA (n = 43). INR were determined using 8 different thromboplastin reagents on the same automated coagulation instrument. Chromogenic factor X, which is supposed to be insensitive to the presence of LA, was also measured. The results are the following: provided INR was calculated using calibrated reference plasmas, there was no significant difference between INR values obtained with the 8 reagents, both in the non-LA and in the LA groups (CV: 5.9 and 6.7%. respectively). Closer examination revealed that INR results obtained with one reagent (the recombinant thromboplastin Innovin) diverged from those of the 7 others, leading to an overestimation of INR, to a very large extent in some instances. However this effect was restricted to a subset of the patient population with LA (6 out of 43). Finally, the relationship between INR (average value obtained using the 8 reagents) and factor X was identical in non-LA and in LA patient groups. We conclude that, provided the reagents which display the LA interference are identified and excluded for this purpose, the INR system is valid for monitoring oral anticoagulant treatment in patients with LA.

Introduction La thrombose portale est une complication frequente de la cirrhose mais son mecanisme n’est pas clairement etabli [1, 2]. Les modifications des tests d’hemostase dans le systeme porte ont ete peu etudiees. Le test de generation de thrombine (TGT) evalue le potentiel endogene de thrombine refletant l’equilibre entre facteurs pro et anticoagulants. Le but de notre etude etait de comparer les tests d’hemostase et en particulier le TGT dans le systeme porte et la circulation veineuse peripherique chez des malades atteints de cirrhose. Patients et Methodes Les tests d’hemostase (Fragment 1+2 de la prothrombine et D-dimeres, Temps de Quick, TCA, dosages des Facteurs II, V et VIII, fibrinogene, taux fonctionnels d’Antithrombine, proteines C et S, TGT) ont ete mesures dans le sang portal et le sang peripherique, preleves de maniere simultanee chez 28 malades atteints de cirrhose a l’occasion de la mise en place d’un TIPS. Le test non parametrique de Wilcoxon etait utilise pour la comparaison des deux territoires. Resultats Dans le sang portal ; le taux du facteur VIII etait correle a la severite de la cirrhose (p = 0,017) et aux taux des proteines C et S. Au TGT, la valeur mediane du rapport de sensibilite a la thrombomoduline du temoin etait de 1,011 (0,78-1,43). Chez la majorite des patients, ces rapports etaient superieurs a 1,6, temoin d’une resistance a la thrombomoduline en faveur d’une hypercoagulabilite equivalente entre les deux circulations (p = 0,274). Cette resistance a la thrombomoduline etait significativement plus importante chez les patients dont l’atteinte hepatique etait plus severe. Conclusion Cette etude confirme que l’augmentation du facteur VIII est correlee a la gravite de la cirrhose. Elle montre que la cirrhose s’accompagne d’un etat d’hypercoagulabilite globale, plus important au niveau portal et correle a sa severite. Elle suggere la realisation d’etudes complementaires pour preciser l’origine de cette hypercoagulabilite. Remerciements, financements, autres Nous remercions le Fond de Recherche de la SNFGE qui a permis le financement de cette etude.

Levels of major parameters of fibrinolysis were measured in 50 normal human fetuses between 19 and 39 weeks of gestation and compared to those of 50 healthy normal pregnant women and 30 adult controls. In fetuses, euglobulin clot lysis time (ECLT) was significantly shortened, plasminogen level was low and histidine-rich glycoprotein undetectable. While t-PA and u-PA levels were slightly lower than in adult controls, a significant decrease in PAI activity was demontrated and no PAI-2 could be detected in fetal plasma. In contrast with these findings, the fibrinolytic equilibrium of pregnant women exhibited a prolonged ECLT and a strong increase in both PAI activity and PAI-2 antigen levels, while only a moderate elevation in u-PA and t-PA levels was measured.

For years, laboratories have used evacuated glass tubes for blood collection. To improve the safety of blood collection, plastic tubes have been developed but various interactions with the coagulation system and/or antithrombotic drugs were reported with the first generation of such tubes. The aim of this multicentre study was to compare the results of different coagulation parameters measured in evacuated polymer (polyethylene terephtalate outer layer and polypropylene inner layer) tubes (Vacuette®, Greiner Bio-One) containing two different citrate concentrations (0.109 M and 0.129 M) with those measured in evacuated siliconized glass tubes (Vacutainer®, Becton-Dickinson) containing the same citrate concentrations. In addition, the impact of aging of the polymer tube was investigated by collecting blood samples in tubes at 11 months (beginning of shelf life=BSL) and at 1 month (end of shelf life=ESF) from expiry (only evaluated using the 0.109 M citrate concentration). A single batch of the different tubes was used in the present study. For that purpose, blood was drawn in the 5 participating centers from untreated patients (n=60), patients on oral anticoagulant treatment (OAT, n=168), and patients treated with either unfractionated heparin (UFH, n=123) or a low molecular weight derivative (LMWH, n=106). The sampling order of the different tubes was randomly defined. Prothrombin time (PT) or INR, activated partial thromboplastin time (APTT), factor V as well as anti-Xa activity (when applicable) were performed in each center using its own routine techniques, immediately after centrifugation. Coagulation activation markers i.e. thrombin-antithrombin complexes (TAT) and prothrombin fragment 1+2 (F1+2) were analyzed in a subset of untreated patients (n=30) after centralization in a single center. In addition, the stability of the samples was evaluated for the routine tests over a 6-hour period in untreated patients. The results of the coagulation tests were significantly different in polymer and glass tubes, from an analytical point of view (paired Student’s T-test). However, the difference was without any clinical relevance (Bland-Altman analysis), whatever the citrate concentration and the patients group. Furthermore, there was no significant difference in the results obtained in BSL and ESL polymer tubes. Interestingly, the plasma levels of TAT and F1+2 were significantly lower when blood was drawn in the polymer tubes for both citrate concentrations, even if the markers remained within the expected range in all cases. A significant evolution of the results, comparable for all types of tubes, was demonstrated for PT, APTT and FV over the 6-hour period. These results suggest that samples collected into the Vacuette® polymer tubes allow accurate routine coagulation testing both in untreated patients and in patients on traditional anticoagulant treatment during the whole shelf life indicated by the manufacturer. In addition, the observation of a lower activation of the coagulation system in the polymer tubes than in the glass tubes, containing a similar citrate concentration, deserves to be further investigated.

This report concerns 34 cases of heparin-associated thrombocytopenia in which standard heparin has been systematically replaced by LMW heparin CY 216 Choay. There were 21 women and 13 men, mean age, 69 years. Twenty-six of the 34 cases had thrombotic complications. All of the patients were treated by standard heparin of porcine mucosal origin, in most cases for prevention of deep vein thrombosis and pulmonary embolism. Twenty-six cases occurred along with orthopedic and traumatologic surgery (especially for total hip replacement, 15 cases). Four cases involved vascular surgery and three involved medical thrombotic disease. Standard heparin treatment was always replaced by LMW heparin CY 216. Initial doses were 0.30 ml three times daily, later increased to obtain global hypocoagulability. Surgical procedures (embolectomy and vena cava filter) were carried out when necessary. Five patients had thrombolytic treatment (urokinase) in conjunction with LMW heparin. The overall results were 31 recoveries, but with seven severe sequelae (three amputations and four hemiplegias) most often attributable to the first ischemic attack, and three deaths. For the last 16 patients, indirect platelet aggregation tests were performed (control platelet-rich plasma plus patient platelet-poor plasma plus LMW heparin): nine cases had negative tests, with nine recoveries and seven cases had positive tests, with five recoveries and two deaths.(ABSTRACT TRUNCATED AT 250 WORDS)

45 patients with pulmonary embolism (PE) were treated by a very low molecular weight heparin fragment (CY 222, Institut CHOAY - France) in an open range dose study. Patients were included into three groups (I, II and III) and received respectively 500, 750 or 1000 IC (Institut Choay) antiXa units/ kg/day by continuous intravenous infusion for ten days. The laboratory screen carried out at Day 0 and at 2 - 8 - 12 - 24 - 36, 48 hours and then every day until Day 10, included : Platelet count, Thromboelastography (TEG) on platelet rich plasma (PRP), Amidolytic assays for anti Xa (CBS 3139 STAGO) and anti Ila (S2238 KABI) activities (Calibration with Hepanorm STAGO), Heptest (Diamed, France), Amidolytic assay for tissue plasminogen activator (t-PA) and its inhibitor (PAI, Verheijen's method). Results showed no modification of platelet count and t-PA or PAI levels; An hypocoagulabi 1 ity was demonstrated with TEG on PRP (r+k > 35 mm) in 20 cases (3 patients from group III with haemorrhagic manifestations and 17 cases from groups II and III without complication). Anti-IIa activities were always lower than 0.15 U/ml. Anti-Xa activities and Heptest (Control adult values = 12.75 sec + 1.15) were (Mean + SD):Any significant biological difference between patients who have good or poor clinical results was observed, excepted for 1 patient who had a high level of PAI at Day 0 and recurrence of PE.

Levels and plasmatic distribution of protein S were studied on umbilical cordplasmas from 25 normal full-term newborns (N) and 15 normal fetuses (F) between20 and 30 weeks of gestation. Samples from fetuses were collected for antenatal diagnosis by direct puncture of the umbilical vein under high resolution real-time ultrasound. Total protein S(PS) level was determined using Laurell rocket immuno-electrophoresis (Diagnostica Stago, Asnifcres-France). Free PS wasmeasured using this latter method, afterprecipitation of C4b-BP-bound-PS by polyethylene glycol (PEG). Normal pool plasma, treated as well, was considered as the reference curve. C4b-binding protein (C4b-BP) determinations were conducted by Laurell rocket immunoelectrophoresis. The qualitative distribution of free PS and C4b-BP-bound-PS in plasma was also assessed by crossed-immunoelectrophoresis(CIE). Results (mean - SD) were expressed in percentage, in relation to healthy adults values (n = 15). Low levels of total PS were obtained in all fetuses (16.4 ±4.2) and newborns (36.4 ±9.5) as compared to adults (91.6 ± 12.2). Free protein S level was also decreased both in fetuses (22.2 ±6.0) and newborns (48.5 ± 12.1 versus 89.4 ± 26.3 in adults). At these stages of development, the ratio Free PS / Total PS (both values were obtained according to a reference curve performed with a normal adult pool plasma untreated by PEG) was significantly higher as compared to normal adults (0.82 ±0.07 in F, 0.64 ±0.17 in N and 0.39 ±0.11 in A, p‹0.001, Student t test). The predominance of free PS was also visualized in the CIE patterns. These data may be explained by undetectable C4b-BP in 21-week old fetuses (‹2% in 10 cases). After the 26th week of gestation C4b-BP level was 7.8 ±7.4 ‹n=5) and reached a value of 19.2 ±15.6 in newborns (adults = 95.7 ±14.7). In human fetus and newborn, PS essentially circulates under free form and this might compensate the decrease of the total PS level.

Levels of total and free Protein S (PS) were measured on plasmas from 28 women during normal (n = 15, mean age = 25) and pathological pregnancy (Hypertension or preeclampsia, n = 13, mean age = 27). Prepartum (PreP) samples were obtained in the third trimester between the 30th and the 40th weeks of gestation, and postpartum (PostP) blood collected in the 5 days after delivery. Total PS level was determined using Laurel 1 rocket immunoelectrophoresis (Diagnostica Stago, Asnifcres-France). Free PS was measured using the same method after precipitation of C4b-BP-Bound-PS by polyethylene glycol. C4b-Binding Protein (C4b-BP) determinations were conducted by Laurel 1 method as well. Results were expressed as a percentage (Mean ± SD) of a normal adult pool (n = 15).A significant decrease of free PS level was observed both in normal or pathological pregnancy and in postpartum. These data might be explained, by the increase of C4b-BP plasmatic level.*= p

Experimental studies on rabbits have shown that CY 222, a low molecular weight heparin (mean: 2.500 daltons), has the anti-thrombosis properties of heparin but reduces the risk of haemorrhage in optimal doses of 1.000 AXa IC (Institut Choay) units/kg/day. The safety and effectiveness of CY 222 were tested in 47 patients presenting with a less than 5 days' old pulmonary embolism. The patients were divided into three groups according to dosage: group I (n = 16) received 500 AXa ICu/kg/day; group II (n = 17), 750 AX ICu/kg/day, and group III (n = 14), 1.000 AXa ICu/kg/day. The drug was administered by continuous intravenous infusion during 10 days. Its effectiveness was assessed from the Miller index calculated on conventional pulmonary angiograms on days 0, 5 and 10. On the 10th day of treatment, the percentage of revascularization was similar in all three groups (group I 65.9 +/- 9.9 p. 100; group II 71 +/- 6.8 p. 100; group III 68 +/- 8.5 p. 100), but the improvement was significantly more rapid in group III patients. Embolism recurred in 5 cases (2 in group I, 1 in group II, 2 in group III) and was fatal in 1 case (group I). Haemorrhagic complications were noted in 3 cases (group III patients). Except for thromboelastography, all coagulation tests were unmodified by CY 222. The anti Xa and the (very low) anti IIa activities of the drug were directly related to the doses administered.(ABSTRACT TRUNCATED AT 250 WORDS)

In a previous open study, a low-molecular-weight-heparinEnoxa-parine in a dose of 40 mg/24 hrs by subcutaneous (SC) injection, had been shown to be efficient and safe in preventing DVT after total hip replacement (THR), for a non traumatic hip disease. 103 patients (mean age : 82 years ± 10, mean weight : 52 kg ± 10) undergoing an orthopedic operation for fracture of femoral neck were included ina randomized, double blind study. These patients receivedSC Enoxaparine according to two different regimens : 54 patients received the treatment A (2 daily injections of 20 mg), 49 patients received the treatment B (1 daily injection of 40 mg).In both groups, administration of 40 mg of Enoxaparine was begun 12 hours before operation.Patients weretreated for 10-15 days, until bilateral ascending phlebography (BAP) had been completed.Lower limbs BAP were performed in 97 patients. The incidence of DVT is low and not significantly different betweenthe two regimens :a proximal DVT was detected in 6 patients of the group A and in 2 patients of the group B. This differenceis not significant(p = 0.28).3 patients of each group hada distal DVT. No clinical pulmonary embolism occurred.There was no serious bleeding complication, and the two groups are not significantly different on this point; 2 patients in each group had an important hematoma of the thigh.One hematoma, in a patient who received the treatment B, required a surgical treatment.Red cell transfusion requirementswere 2.6U+1.8 in the group A and 2.5 U ±1.4 in the group B (p = 0.84)There was no significant difference in the daily hemoglobinlevels between the two groups.This study shows that one daily SC injection of 40 mg of Enoxaparine is as efficient as two daily SC injections of20 mg of Enoxaparine in preventing DVT, in very elderly patients undergoing orthopedic operation for fracture of the femoral neck. The frequencies of bleeding complications in each group were not significantly different.Enoxaparine (LOVENOXR) - PHARMUKA S.F.

The Authors report a new iatrogenic complication of preventive or curative standard heparinotherapy. This so called "white clot syndrome" or "Heparin associated thrombocytopenia and thrombosis" is an anatomico-clinical entity characterized by severe, multiple, recurrent, arterial and/or venous thromboembolic accidents which are sometimes fatal. They appear, paradoxically, under heparin treatment and are concomitant with thrombocytopenia (generally less than 100 10(9)/l). Antiplatelet immunoallergic phenomena, via type IgG antibodies, induced by heparin, are the most commonly admitted in physiopathology; however they remain controversial. Treatment of "WCS" includes urgent suppression of standard heparin, administration of antivitamin K, or the new generation of low molecular weight heparin. This series presents 26 cases of HATT treated by LMWH (CY 216 CHOAY). Severe complications must be surgically cured. Prevention of WCS or HATT is mandatory by the systematic survey of platelet count during heparinotherapy.

Levels and plasma distribution of protein S were measured on umbilical cord plasmas from 25 normal full-term newborns and 25 normal fetuses which were between 20 and 31 weeks of gestation. Fetal blood samples were obtained by direct puncture of the umbilical vein under high resolution real-time ultrasound. Total and Free protein S levels were found to be lower in all fetuses and newborns as compared to normal adults. The calculated ratio Total protein S/Free protein S and crossed immunoelectrophoresis patterns indicate that protein S circulates essentially in the free form in fetuses and newborns. These data may be explained by the low levels of C4b-binding protein observed at these stages of development.

A systematic coagulation study of forty different species of vertebrates using autoreagents (i.e: prepared from their own tissues), revealed biological activity of factors thought till now to be absent so early in the phylogenetic evolution because of their unreactivity with respect to standard reagents used currently in man.Hybrid plasma fractions from man and other animal species demonstrated that the best extrinsic pathway kinetics were obtained by associating specific thromboplastins and specific factors VII-X.The comparison of extraphysiological coagulation activators (such as Russell viper venom, Echis carinatus viper venom, staphylocoagulase, thrombin coagulase, Bothrops atrox viper venom, Agkistrodon contortrix snake venom) permits a chronological approximation of the coagulation factors evolutionary variations.It should be noted that the Russell viper venom pathway is found in all vertebrates.

Experimental studies showed that CY 222 (MW 2500 - 250 U AXAIC/ mg - 25 Ul/mg) kept the profibrinolytic properties of heparin with a considerable reduction of bleeding risk and that the optimal dose was about 1 000 U AXAIC/kg/day in rabbits. We have tested the dose related effectiveness and tolerance of this drug in 47 patients (pts) with acute pulmonary embolism (PE) less than 5 days. Pts were divided into 3 groups receiving : Group I : 500 (n = 16), Group II : 750 (n = 17), Group III : 1 000 (n = 14) U AXAIC/kg/day by continuous intravenous infusion for ten days. Effectiveness was appreciated by Miller’ s index with pulmonary angiography (PA) performed before, on the 5th day and on the 10 th day of treatment. The decrease of Miller’ s index mean value was comparable in the 3 groups. But the improvement was faster in the group IIIWe noticed 5 recurrent PE (2 in Group I, 1 in Group II and 2 in Group III) of which one was fatal (Group I), and 2 hematoma at the venous punction site in Group III.In conclusion, CY 222 is a safe treatment of PE and the most effective dose seems to be 1 000 U AXA IC/kg/day in spite of minor bleeding.

This study involved two stages: In vitro study. Three LMWHs, CY 216-CY 222 (Choay) and PK 10169 (Pharmuka), were compared to standard heparin, SH (Choay), and to a high-molecular-weight heparin, HAF (Choay), which particularly revealed a dissociation of anti-Xa (++) and anti-IIa (+) activities for the LMWHs: (table: see text) In vivo study. Healthy volunteers. Subcutaneously injections of PK 10169 or CY 222 according to various protocols (rythm of injections, dosage). For both LMWHs, the dissociation of anti-Xa and anti-IIa activities reoccurs in vivo. A peak of anti-Xa effect was observed 3 to 4 hours after the injections. The duration period of this activity is about 12 hours. The importance of the global platelet tests HT and TEG (modified for the highest doses) should be noted. Study of haemorrhagic tendencies in patients with slight thrombotic risk. From this preliminary study, protocols with CY 216, CY 222 and PK 10169 are proposed for the prevention of thrombotic risk in orthopaedic surgery (injections at 12-hour intervals).