European registry of babies born to mothers with antiphospholipid syndrome.

OBJECTIVES: This study aimed to describe the long-term outcome and immunological status of children born to mothers with antiphospholipid syndrome, to determine the factors responsible for childhood abnormalities, and to correlate the child's immunological profile with their mothers. METHODS: A prospective follow-up of a European multicentre cohort was conducted. The follow-up consisted of clinical examination, growth data, neurodevelopmental milestones and antiphospholipid antibodies (APL) screening. Children were examined at 3, 9, 24 months and 5 years. RESULTS: 134 children were analysed (female sex in 65 cases, birth weight 3000±500 g, height 48±3 cm). Sixteen per cent had a preterm birth (<37 weeks; n=22), and 14% weighted less than 2500 g at birth (n=19). Neonatal complications were noted in 18 cases (13%), with five infections (4%). During the 5-year follow-up, no thrombosis or systemic lupus erythematosus (SLE) was noted. Four children displayed behavioural abnormalities, which consisted of autism, hyperactive behaviour, feeding disorder with language delay and axial hypotony with psychomotor delay. At birth lupus anticoagulant was present in four (4%), anticardiolipin antibodies (ACL) IgG in 18 (16%), anti-ß(2) glycoprotein-I (anti-ß2GPI) IgG/M in 16 (15%) and three (3%), respectively. ACL IgG and anti-ß2GPI disappeared at 6 months in nine (17%) and nine (18%), whereas APL persisted in 10% of children. ACL and anti-ß2GPI IgG were correlated with the same mother's antibodies before 6 months of age (p<0.05). CONCLUSION: Despite the presence of APL in children, thrombosis or SLE were not observed. The presence of neurodevelopmental abnormalities seems to be more important in these children, and could justify long-term follow-up.

Laboratory criteria of the obstetrical antiphospholipid syndrome. Data from a multicentric prospective European women cohort.

A debate on updating the laboratory criteria of antiphospholipid syndrome (APS) was recently opened in view to lower the risk of over diagnosis of the syndrome. Based on data related to thrombotic APS, it proposes the exclusion of anticardiolipin antibodies (aCL) and anti-beta2-glycoprotein 1 (a-beta2-GPI) IgM detection. Here, we examine this possibility in a study which focuses on obstetrical APS (OAPS). We report new data on a prospective multicenter European cohort of 109 pregnant women having APS. Among them, 73 had purely obstetrical APS, not associated to autoimmune diseases or thrombosis. Isolated antibodies and isolated aCL positivity were present in 50/109 (46%) and in 34/109 (31%) of the women, respectively. An isolated a-beta2-GPI IgM was present in three women. These results suggest that aCL and a-beta2-GPI IgM cannot be dropped for the diagnosis and classification of OAPS. The low level of some antibodies associated with severe obstetrical complications raise the issue of keeping or not the same laboratory criteria for OAPS and for thrombotic APS and whether additional criteria after large prospective studies could further improve diagnosis.

Diluted Russell viper-venom time improves identification of antiphospholipid syndrome in a lupus anticoagulant-positive patient population.

The objective of this retrospective study was to evaluate the potential ability of diluted Russell viper-venom time (dRVVT) to identify antiphospholipid syndrome (APS) in a lupus anticoagulant (LA)-positive patient population, already selected by other LA clotting tests. Our cohort of positive LA patients was first identified in our outpatients population by the following sensitive LA-detecting tests: Rosner index, diluted prothrombin time (dPT) and Rosove index. Then the 227 consecutive LA-positive patients were tested for dRVVT with the same blood sample. Anticardiolipin (aCL) and anti-beta(2)-glycoprotein-I (beta(2)GPI) autoantibodies assays were also performed. APS using Sapporo clinical criteria revised at Sydney, was found in 116 of these 227 consecutive LA-positive patients. Results of the different tests were analysed statistically. Using univariate analysis, dRVVT, dPT, IgG aCL and IgG anti-beta(2)GPI autoantibodies were significantly associated with APS. The receiver operating-characteristics (ROC) curve defined the best cut-off value for dRVVT ratio at 1.61 with a good specificity (78%) and a lower sensitivity (53%). A multivariate analysis using a binary logistic procedure, retained the dRVVT ratio (> or = 1.61) and IgG anti-beta(2)GPI autoantibodies (> 15 USG) as being associated with APS (p = 0.018; odds ratio [OR] 2.39; 95% confidence interval [CI] 1.2-4.7, and p = 0.0001; OR 3.2; 95% CI 1.5-6.5, respectively). To conclude, these results agree with the need for LA criteria favouring specificity over sensitivity. The use of a threshold around 1.6 for dRVVT ratio should help discriminate APS from non-APS patients.

Antiphosphatidylethanolamine antibodies are associated with an increased odds ratio for thrombosis. A multicenter study with the participation of the European Forum on antiphospholipid antibodies.

A multicenter study was set up to evaluate the prevalence, clinical and biological significance of antiphosphatidylethanolamine antibodies (aPE) in thrombotic patients with or without the main known clinical and biological risk factors for thrombosis. APE and antibodies, defined as the laboratory criteria of antiphospholipid syndrome (APS) -lupus anticoagulant, anticardiolipin and anti-beta(2)-GPI antibodies were measured in 270 patients with thrombosis (234 venous and 37 arterial) and 236 matched controls. APE were found in 15% of thrombotic patients compared to 3% of controls (p < 0.001) with no predominant isotype, no association with the main known clinical or biological risk factors for thrombosis neither with a type of thrombosis, arterial or venous. In a multivariate logistic regression analysis of antibodies, aPE showed the highest association with thrombosis (odds ratio [OR]: 4.2, p < 0.001). Moreover, using a multivariate analysis in a case-control subgroup study on 158 patients, IgGaPE were found to be significantly associated with venous thrombosis (OR:6;p = 0.005). Interestingly, 25 of the 40 aPE-positive patients (63%) were negative for the APS laboratory criteria. Most of them (21/25) had venous thrombosis, recurrent in ten of them. Four patients also suffered from early or late miscarriages. Our results underline the strength of the association between the presence of aPE and thrombosis and suggest their measurement in thrombotic patients, especially when lupus anticoagulant, anticardiolipin or anti-beta(2)-GPI antibodies are absent.

V meeting of the European forum on antiphospholipid antibodies.

The "V Meeting of the European Forum on Antiphospholipid Antibodies" was organized in Barcelona, Catalonia, Spain, on December 2-3, 2005. This biannual meeting becomes a real forum for the interchange of information on current research on the field antiphospholipid antibodies and the antiphospholipid syndrome, as well as a starting point for many new research projects. About 170 physicians and researchers from a great variety of medical specialities (internists, rheumatologists, hematologists, obstetricians, pediatricians, neurologists, cardiologists, immunologists, and biologists, among others) coming from 20 European countries attended the sessions. This report summarizes the main studies and new research projects presented during this Forum Meeting.

Variability of anti-beta2 glycoprotein I antibodies measurement by commercial assays.

The aim of this study was to evaluate the agreement in assay results between commercial kits for the measurement of anti-beta2glycoprotein 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.

Inter-laboratory variability of anti-beta2-glycoprotein I measurement. A collaborative study in the frame of the European Forum on Antiphospholipid Antibodies Standardization Group.

Inter-laboratory variability of anti-beta2-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-beta2-glycoprotein I measurements between centers, the agreement on results with high- and medium-positive samples was good.

Minimal requirements for antiphospholipid antibodies ELISAs proposed by the European Forum on antiphospholipid antibodies.

Antiphospholipid ELISAs are part of the Antiphospholipid Antibodies Syndrome classification criteria, having the same diagnostic value as lupus anticoagulant. However, sometimes their results appear scarcely meaningful especially when wide metanalyses studies are performed, probably because of their well-known inter-laboratory variability. The application of a common protocol was shown to improve the test reproducibility, but this observation did not have any influence on the routine performances. After discussion among experts at the European level, we identified four conditions named "minimal requirements" considered useful to decrease the inter-laboratory variability: (1) to run the samples in duplicate; (2) to determine the cut off level in each laboratory analysing at least 50 samples from normal subjects, possibly age- and sex-matched with the patient population usually attending the Centre; (3) to calculate the cut-off level in percentiles; (4) to use stable external control in the tests. A collaborative study involving 36 European centres proved that the use of monoclonal anti-beta2 glycoprotein I antibodies, HCAL (IgG) and EY2C9 (IgM) as standards, can help to reduce the inter-laboratory coefficient of variation both in anticardiolipin (aCL) and anti-beta2GPI (anti-beta2 glycoprotein I) ELISA. Therefore, we propose HCAL and EY2C9 as external controls, but other monoclonal or polyclonal preparations may be considered. During an interactive workshop held last May in Italy, 16 companies producing these tests agreed to consider the introduction of the "requirements" in their products. We suggest to adopt these "requirements" particularly in clinical studies, in order to compare more easily the literature data.

Outcomes and treatment of obstetrical antiphospholipid syndrome in women with low antiphospholipid antibody levels.

Our objective was to determine whether there is a relationship between low antiphospholipid (aPL) antibody levels and the obstetrical complications of antiphospholipid syndrome (APS) and to analyze the impact of conventional APS treatment in patients with low aPL levels. To this end, we retrospectively reviewed the files of all patients referred to our unit (2003-2010) for unexplained pregnancy morbidity, with an aPL test result. We compared patients with APS confirmed by Sapporo criteria (Group 1) with patients with APS-like obstetrical complications with an aPL titer below the intermediate titer (Group 2). Overall, 57 patients were included (25 in Group 1; 32 in Group 2). Obstetrical events were recurrent spontaneous abortion <10th week of gestation (n=9 patients in Group 1; n=13 patients in Group 2), fetal death (n=11 and 16, respectively), preeclampsia (n=5 in Group 1; n=6 in Group 2). The total number of obstetrical events per patient was very similar before APS treatment (3 [1-8] in Group 1; 3 [1-6] in Group 2) and decreased significantly after APS treatment to 0 [0-2] and 0 [0-2], respectively (p<0.05). The incidence of premature births and the characteristics of neonates were similar in the two groups. In this study, treatment of patients with low aPL levels and APS-like obstetrical events was associated with outcomes similar to those found in otherwise normal women with recurrent miscarriage or other adverse events. However, properly designed treatment trials would be required to prove the benefit of such treatments.

Anti-platelet factor 4/heparin antibodies from patients with heparin-induced thrombocytopenia provoke direct activation of microvascular endothelial cells.

Heparin-induced thrombocytopenia (HIT) is a serious complication that occurs in approximately 1-5% of patients treated with heparin and may be associated with severe thrombotic events. HIT is mediated by antibodies directed mostly to epitope(s) formed by complexes between heparin or other anionic mucopolysaccharides and platelet factor 4 (PF4). Anti-PF4/heparin IgG antibodies from six patients with HIT were affinity purified and assessed for interaction with human microvascular and macrovascular endothelial cells (EC). The antibodies directly activated primary cultures of human bone marrow microvascular EC (HBMEC) and SV40 immortalized HBMEC (TrHBMEC) only in the presence of PF4, but did not activate macrovascular human umbilical vein EC (HUVEC) under the same conditions. These antibodies were found to bind to TrHBMEC through the F(ab)(2) portion of the anti-PF4/heparin IgG. TrHBMEC activation was characterized by an augmented release of IL-6, von Willebrand factor, soluble thrombomodulin, and by an elevated expression of the adhesion molecules P-selectin, E-selectin and vascular cellular endothelial molecule-I to different degrees. Enhanced monocyte adhesion to PF4/heparin antibody-treated TrHBMEC (33-72% adhesion) was also observed. None of these effects occurred with unstimulated HUVEC. However, pre-treatment of HUVEC with tumor necrosis factor-alpha resulted in the same changes observed with microvascular EC exposed to the HIT antibodies. Our findings indicate that anti-PF4/heparin antibodies directly activate microvascular EC while interaction with macrovascular EC requires pre-activation. These results may explain some of the specific clinical manifestations in HIT.

Antiphospholipid syndrome: clinical and immunologic manifestations and patterns of disease expression in a cohort of 1,000 patients.

OBJECTIVE: To analyze the clinical and immunologic manifestations of antiphospholipid syndrome (APS) in a large cohort of patients and to define patterns of disease expression. METHODS: The clinical and serologic features of APS (Sapporo preliminary criteria) in 1,000 patients from 13 European countries were analyzed using a computerized database. RESULTS: The cohort consisted of 820 female patients (82.0%) and 180 male patients (18.0%) with a mean +/- SD age of 42 +/- 14 years at study entry. "Primary" APS was present in 53.1% of the patients; APS was associated with systemic lupus erythematosus (SLE) in 36.2%, with lupus-like syndrome in 5.0%, and with other diseases in 5.9%. A variety of thrombotic manifestations affecting the majority of organs were recorded. A catastrophic APS occurred in 0.8% of the patients. Patients with APS associated with SLE had more episodes of arthritis and livedo reticularis, and more frequently exhibited thrombocytopenia and leukopenia. Female patients had a higher frequency of arthritis, livedo reticularis, and migraine. Male patients had a higher frequency of myocardial infarction, epilepsy, and arterial thrombosis in the lower legs and feet. In 28 patients (2.8%), disease onset occurred before age 15; these patients had more episodes of chorea and jugular vein thrombosis than the remaining patients. In 127 patients (12.7%), disease onset occurred after age 50; most of these patients were men. These patients had a higher frequency of stroke and angina pectoris, but a lower frequency of livedo reticularis, than the remaining patients. CONCLUSION: APS may affect any organ of the body and display a broad spectrum of manifestations. An association with SLE, the patient's sex, and the patient's age at disease onset can modify the disease expression and define specific subsets of APS.

Signes toxiques, signes pathogenetiques et composition des venins de serpents / Toxic and pathogenetics signs and composition of snakes venoms

La connaissancedes venins de serpents montre que les signes toxiques comme les signes pathogenetiques sont dus aux differents constituants du venin analyse. Il est interessant de faire le rapprochement entre signes cliniques et biologiques, et composition du venin, mais ce rapprochement ne peut etre que tres imparfait car les substances contenues dans chaque venin ne sont pas encore toutes connues, quelques unes seulement ayant ete recherchees. La reparrtition des multiples constituants de venin d'innombrables serpents est d'une diversite infinite. L'etude de la composition et de l'effet des venins incite a faire de nouvelles pathogenesies ou a refaire des pathogenesies a doses infinitesimal;es sur le plan clinique et biologique. En effetmsile merveilleux outil therapeutique qu'est Lachesis est bien connu, nous utilisons aussi Naja naja, Vepera, Bothrops, Crotalus, mais sans connaitre toutes les possibilites d'application qu'ils offrent car les signes pathogenetiques sont soit mal connus, soit mal explores, leseffets connus ayant ete mesures a doses subtoxiques ou toxiques. Nous pourrions aussi agrandir considerablement l'eventail therapeutique en y faisant rentrer d'autres venins de serpents tout aussi utiles