Management of the thrombotic risk associated with COVID-19: guidance for the hemostasis laboratory.

Coronavirus disease 2019 (COVID-19) is associated with extreme inflammatory response, disordered hemostasis and high thrombotic risk. A high incidence of thromboembolic events has been reported despite thromboprophylaxis, raising the question of a more effective anticoagulation. First-line hemostasis tests such as activated partial thromboplastin time, prothrombin time, fibrinogen and D-dimers are proposed for assessing thrombotic risk and monitoring hemostasis, but are vulnerable to many drawbacks affecting their reliability and clinical relevance. Specialized hemostasis-related tests (soluble fibrin complexes, tests assessing fibrinolytic capacity, viscoelastic tests, thrombin generation) may have an interest to assess the thrombotic risk associated with COVID-19. Another challenge for the hemostasis laboratory is the monitoring of heparin treatment, especially unfractionated heparin in the setting of an extreme inflammatory response. This review aimed at evaluating the role of hemostasis tests in the management of COVID-19 and discussing their main limitations.

Immunogenicity, efficacy and safety of Nuwiq® (human-cl rhFVIII) in previously untreated patients with severe haemophilia A-Interim results from the NuProtect Study.

INTRODUCTION: Nuwiq® (Human-cl rhFVIII) is a fourth generation recombinant FVIII, produced in a human cell line, without chemical modification or protein fusion. No inhibitors developed in studies with Nuwiq® in 201 previously treated patients with haemophilia A (HA). The immunogenicity, efficacy and safety of Nuwiq® in previously untreated patients (PUPs) with severe HA are being assessed in the ongoing NuProtect study. METHODS: The study, conducted across 38 centres worldwide, is evaluating 110 true PUPs of all ages and ethnicities enrolled for study up to 100 exposure days (EDs) or 5 years maximum. The primary objective is to assess the immunogenicity of Nuwiq® (inhibitor activity ≥0.6 BU) using the Nijmegen-modified Bethesda assay at a central laboratory. RESULTS: Data for 66 PUPs with ≥20 EDs from a preplanned interim analysis were analysed. High-titre (HT) inhibitors developed in 8 of 66 patients after a median of 11.5 EDs (range 6-24). Five patients developed low-titre inhibitors (4 transient). The cumulative incidence (95% confidence interval) was 12.8% (4.5%, 21.2%) for HT inhibitors and 20.8% (10.7%, 31.0%) for all inhibitors. During inhibitor-free periods, median annualized bleeding rates during prophylaxis were 0 for spontaneous bleeds and 2.40 for all bleeds. Efficacy was rated as "excellent" or "good" in treating 91.8% of bleeds. Efficacy of surgical prophylaxis was "excellent" or "good" for 8 (89%) procedures and "moderate" for 1 (11%). No tolerability concerns were evident. CONCLUSION: These interim data show a cumulative incidence of 12.8% for HT inhibitors and convincing efficacy and tolerability in PUPs treated with Nuwiq® .

Postauthorization safety study of Clottafact® , a triply secured fibrinogen concentrate in acquired fibrinogen deficiency: a prospective observational study.

BACKGROUND AND OBJECTIVES: A postauthorization safety study was performed between 2009 and 2012 to describe the use of Clottafact® in acquired fibrinogen deficiency in real-life medical practice in France. MATERIALS AND METHODS: One hundred and fifty patients were planned for 28 days of prospective follow-up after infusion. The analysis of this observational study was descriptive and performed according to the type of treatment (curative or preventive) and the origin of the bleed. RESULTS: One hundred and fifty-six patients (16-87 years) were included in 13 centres and treated in five different medical bleeding situations: postpartum (59), other gynaecological/obstetrical (6), trauma (34), liver (13), cardiovascular (23) and other various bleeding situations (21). The mean follow-up time was 18·9 ± 12·3 days. Two patients presented adverse drug reactions: one a pulmonary embolism and the other a four-site venous thromboembolic episode. All were serious with a dubious causal relationship with the study treatment. Efficacy data were collected as a secondary objective. In 150 patients receiving curative treatment, 117 of 159 infusions (73·6%) were considered as successful by the investigators, 35 as moderate (22%) and seven as no response (4·4%). CONCLUSION: The Clottafact® safety profile observed during the study matched the known profile of fibrinogen during use.

Intravenous thrombolysis for acute ischaemic stroke in patients on direct oral anticoagulants.

BACKGROUND AND PURPOSE: Whereas intravenous thrombolysis (IVT) is allowed for acute ischaemic stroke in patients on vitamin K antagonists with international normalized ratio ≤1.7, there are no similar recommendations for patients on direct oral anticoagulants (DOACs), notably due to the lack of coagulation tests to assess the therapeutic effects. Although the literature is scarce, consisting of small case series and retrospective studies, considering the frequency of this situation the French Vascular Neurology Society and the French Study Group on Haemostasis and Thrombosis have worked on a joint position paper to provide a practical position regarding the emergency management of ischaemic stroke in patients on DOACs. METHOD: Based on a review of the literature, the authors wrote a first text that was submitted to a broad panel of members from the two societies. The text was then amended by the authors to address experts' comments and to reach a consensus. RESULTS: In patients with normal renal function and who stopped the DOAC for at least 48 h, the management should not differ from that in patients without oral anticoagulant. In patients who are still on DOACs, mechanical thrombectomy is encouraged preferentially when applicable in first line. Otherwise, when specific tests are available, values <50 ng/ml indicate that IVT is allowed. In the absence of specific tests, standard tests (thrombin time, prothrombin time and activated partial thromboplastin time) can be used for dabigatran and rivaroxaban, although interpretation of these tests may be less reliable. In some patients on dabigatran, idarucizumab may be used before IVT. CONCLUSIONS: In this expert opinion paper, it is suggested that IVT can be performed in patients selected according to the time elapsed since the drug was last taken, renal function, type of hospital where the patient is admitted and plasma concentration of DOAC.

Post-authorization safety study of Clottafact® , a triply secured fibrinogen concentrate in congenital afibrinogenemia. A prospective observational study.

BACKGROUND AND OBJECTIVES: A new fibrinogen concentrate Clottafact® was developed according to European guidelines on plasma-derived products. A post-authorization safety study was set up in 2009 as part of the risk management plan. This was a non-interventional, prospective, non-comparative, multicenter study of the use of fibrinogen concentrate for congenital afibrinogenemia in real-life medical practice in France. MATERIALS AND METHODS: The analysis was descriptive and performed on 3 subgroups: prophylaxis vs. on-demand treatment, age (<6, <12 and ≥12) and severity of the deficiency. RESULTS: Fourteen patients [1-78 years] were included in 7 centres and followed for 1 year. Twenty-one adverse drug reactions (ADRs) classically reported with fibrinogen (pallor, chills, cough, vomiting, headache, urticaria and erythematous rash) were reported in 5 of 14 patients. Two ADRs were serious: an anaphylactic shock and a subclavian venous thrombosis with a favourable outcome without sequelae. In the nine patients under prophylaxis, 365 of 367 infusions were considered as successful (99·5%) and 2 as failures. For the five patients treated on-demand, the efficacy was rated as excellent for 27 of 48 infusions and good for the 21 others. CONCLUSION: This study confirms that the benefit/risk balance for this fibrinogen concentrate is favourable.

Use of Haemate(®) P as immune tolerance induction in patients with severe haemophilia A who failed previous induction attempts: a multicentre observational study.

Immune tolerance induction (ITI) can eliminate factor VIII (FVIII) inhibitory antibodies that appear during FVIII replacement therapy. If first-line ITI fails, switching to a different FVIII concentrate, especially one containing von Willebrand factor (VWF), has been advocated. The objective of the study was to assess the efficacy and safety of Haemate(®) P, a plasma-derived FVIII concentrate containing high levels of VWF, as ITI in severe haemophilia A patients who had failed at least one prior ITI attempt with a different FVIII concentrate. In this multicentre, observational study, Haemate(®) P was administered at a starting dose of 83-308 IU kg(-1) day(-1) (1500-6000 IU day(-1)). Efficacy was assessed by standard criteria (e.g. Bethesda titre, FVIII recovery and half-life), and bleeding characteristics. Nine patients from six haemophilia centres were treated with Haemate(®) P after failing one (n = 2), two (n = 5) or three (n = 2) prior ITI courses. The median time from inhibitor detection to Haemate(®) P treatment was 5.4 years. The median Haemate(®) P dose was 134 IU kg(-1), and the median treatment duration 32 months. During median of 47 months of follow-up, complete response, partial response and treatment failure were observed in one, three and five patients respectively. Five patients experienced seven adverse events (AEs), including two serious AEs (sepsis). Haemate(®) P was discontinued due to an AE in one patient with a partial response. Haemate(®) P salvage ITI resulted in complete or partial tolerization in four of nine patients (44%) who had failed previous ITI attempts using different FVIII concentrates.

Further characterization of the thrombasthenia-related idiotype OG. Antiidiotype defines a novel epitope(s) shared by fibrinogen B beta chain, vitronectin, and von Willebrand factor and required for binding to beta 3.

A patient (OG) with Glanzmann thrombasthenia became refractory to platelet transfusion after the production of an immunoglobulin G (IgG) isoantibody (Ab1) specific for the integrin subunit beta 3. To determine the frequency at which the OG idiotype is found in the general population and in immune-mediated disease states, we developed a rabbit polyclonal antibody (Ab2) specific for affinity-purified OG anti-beta 3 Fab. The binding of Ab2 to Ab1 is inhibited by purified alpha IIb beta 3. Ab2 als binds to IgG specific for alpha IIb beta 3 obtained from one nonrelated Glanzmann thrombasthenia patient ES who has developed isoantibodies of similar specificity. On the other hand, Ab2 does not recognize alpha IIb beta 3-specific antibodies produced by two Glanzmann thrombasthenia patients, AF and LUC, who have developed isoantibodies with specificities distinct from that of the OG isoantibody. Moreover, Ab2 does not recognize alpha IIb beta 3-specific antibodies developed by three representative patients with (autoimmune) thrombocytopenic purpura or six representative patients with alloimmune thrombocytopenias, nor does it bind to IgG from any of 13 nonimmunized individuals. We have found that Ab2 also binds to selected protein ligands of alpha IIb beta 3 namely, fibrinogen, vitronectin, and von Willebrand factor, but not to other protein ligands or control proteins, such a fibronectin, type I collagen, and albumin. The epitope(s) recognized by Ab2 on each adhesive protein are either very similar or identical since each protein can inhibit the binding of Ab2 to any of the other proteins. The epitope on fibrinogen recognized by Ab2 resides in the B beta chain, and is likely contained within the first 42 amino acids from the NH2 terminus. Since OG IgG inhibits fibrinogen binding to alpha IIb beta 3, the specificity of the OG idiotype defines a novel binding motif for the integrin alpha IIb beta 3 that is shared by fibrinogen, vitronectin, and von Willebrand factor, but distinct from previously described RGD-containing sites on the fibrinogen, A alpha chain or the fibrinogen gamma chain COOH-terminal decapeptide site. Our findings reported here represent an excellent example of molecular mimicry in which an antigen-selected, IgG inhibitor of alpha IIb beta 3 function shares a novel recognition sequence common to three physiologic protein ligands of that receptor.

[Intra-alveolar pulmonary haemorrhage, an unusual manifestation of the primary antiphospholipid syndrome]. / L'hémorragie intra-alvéolaire, une complication rare du syndrome primaire des antiphospholipides.

Pulmonary embolism is the main pulmonary manifestation of primary antiphospholipid syndrome. Other pulmonary manifestations including intra-alveolar haemorrhage are less common. We report a 36-year-old man with a primary antiphospholipid syndrome who presented with an acute respiratory failure due to the association of pulmonary embolism and intra-alveolar haemorrhage. This diagnosis should be systematically considered as it is life threatening and requires a specific therapy.

[Risk factors for thromboembolic disease in young women-the role of hormones]. / Facteurs de risque de maladie veineuse thromboembolique chez les jeunes femmes en contexte hormonal: Risque de survenue d'une maladie thromboembolique veineuse chez les membres de la famille au 1er degré de femmes ayant une maladie veineuse thromboembolique en contexte hormonal : étude « FIT ­ H ¼.

Hormonal exposure in young women increases the risk of venous thromboembolic disease (VTE). Thrombophilia testing is often proposed in women of childbearing age before the initiation of contraception. However, the presence of a familial history of VTE has the potential to be more accurate than the presence of inherited thrombophilia. OBJECTIVE: To demonstrate an association between the risk of VTE in young women with hormonal exposure (pregnancy or oral contraceptive use) and the presence of a previous episode of VTE in their first-degree relatives, according to whether or not a detectable inherited thrombophilia was present. METHODS: We will perform a multicenter case-control cross-sectional study. The main risk factor is defined by the presence of a symptomatic VTE in young women with hormonal exposure. The principle variable is the presence of an objectively diagnosed episode of VTE in first-degree relatives. We will need to include 2,200 family members in 440 cases. EXPECTED RESULTS: We expect to improve understanding of the thrombotic risk in first-degree relatives of patients in hormonal context with or without a past history of VTE.

Management of antiplatelet therapy for non elective invasive procedures of bleeding complications: proposals from the French working group on perioperative haemostasis (GIHP), in collaboration with the French Society of Anaesthesia and Intensive Care Medicine (SFAR).

The French Working Group on Perioperative Haemostasis (GIHP) and the French Study Group on Haemostasis and Thrombosis (GFHT) in collaboration with the French Society of Anaesthesia and Intensive Care Medicine (SFAR) drafted up-to-date proposals on the management of antiplatelet therapy for non-elective invasive procedures or bleeding complications. The proposals were discussed and validated by a vote; all proposals could be assigned with a high strength. Emergency management of oral antiplatelet agents (APA) requires knowledge on their pharmacokinetic/pharmacodynamics parameters, evaluation of the degree of the alteration of haemostatic competence and the associated bleeding risk. Platelet function testing may be considered. When APA-induced bleeding risk may worsen the prognosis, measures should be taken to neutralise antiplatelet therapy by considering not only the efficacy of available means (which can be limited for prasugrel and even more for ticagrelor) but also the risks that these means expose the patient to. The measures include platelet transfusion at the appropriate dose and haemostatic agents (tranexamic acid; rFVIIa for ticagrelor). When possible, postponing non-elective invasive procedures at least for a few hours until the elimination of the active compound (which could compromise the effect of transfused platelets) or if possible a few days (reduction of the effect of APA) should be considered.

[Prolongation of anti vitamin K treatment for 18 months versus placebo after 6 months treatment of a first episode of ideopathic pulmonary embolism: a mutlicentre, randomised double blind trail. The PADIS-EP Trial]. / Prolongation d'un traitement par antivitamine K pendant dix-huit mois versus placebo au décours d'un premier épisode d'embolie pulmonaire idiopathique traité six mois: un essai randomisé multicentrique en double aveugle. Essai "PADIS-EP".

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).

Prospective evaluation of the '4Ts' score and particle gel immunoassay specific to heparin/PF4 for the diagnosis of heparin-induced thrombocytopenia.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is a severe disease that is often difficult to diagnose. A clinical scoring system, the '4Ts' score, has been proposed to estimate its probability before laboratory testing, and a particle gel immunoassay (H/PF4 PaGIA) has also been developed for rapid detection of HIT antibodies. AIM: To evaluate the performance of both methods when HIT is suspected clinically. METHODS: Two hundred thirteen consecutive patients were included in four centers. The probability of HIT was evaluated using the 4Ts score blind to antibody test results. HIT was confirmed only when the serotonin release assay (SRA) was positive. RESULTS: The risk of HIT was evaluated by the 4Ts score as low (LowR), intermediate (IR) or high (HR) in 34.7%, 60.6% and 4.7% of patients, respectively. The negative predictive value (NPV) of the 4Ts score was 100%, as the SRA was negative in all LowR patients. PaGIA was negative in 176 patients without HIT (99.4%, NPV) and the negative likelihood ratio (LR-) was 0.05. PaGIA was positive in 37 patients, including 21 with HIT (positive predictive value = 56.8%), with a positive LR of 11.4. A negative PaGIA result decreased the probability of HIT in IR patients from 10.9% before assay to 0.6%, whereas a positive result did not substantially increase the likelihood for HIT. CONCLUSION: The use of the 4Ts score with PaGIA appears to be a reliable strategy to rule out HIT.

Heparin-induced thrombocytopenia: a frequent complication after cardiac surgery.

Thrombocytopenia is a common problem in cardiovascular patients, and heparin-induced thrombocytopenia (HIT) is therefore frequently suspected. Unfractionated heparin during cardiopulmonary bypass is particularly immunogenic as 25% to 50% post-cardiac surgery patients develop heparin-dependent antibodies but only 1 to 3% will develop HIT. These antibodies recognize a 'self protein', platelet factor 4 (PF4), bound to heparin. Antibodies associated with a high risk of HIT are mainly IgG1 which strongly activate platelets and coagulation, thereby causing thrombocytopenia and thrombosis. A biphasic evolution of platelet count with a secondary decrease after a previous increase following CPB or non-recovery of thrombocytopenia within 6 days post-operatively always requires screening for HIT antibodies. Both functional (platelet activation tests) and immunologic assays (antigen assays) are necessary in every patient to establish the diagnosis of HIT. When the clinical probability of HIT is high, the first requirement is to discontinue heparin, without waiting for results of laboratory investigations. An alternative anticoagulant such as danaparoid sodium (Orgaran) or lepirudin (Refludan) must then be administered since heparin withdrawal alone is insufficient to control the prothrombotic state associated with HIT. The risk of HIT will probably soon decrease due to the wider use of fondaparinux, which does not interact in vitro with PF4, but it could remain significant in patients undergoing cardiac surgery with CPB.

[Neonatal arterial ischemic stroke: Which thrombotic biological risk factors to investigate and which practical consequences?] / Accident vasculaire cérébral ischémique du nouveau-né : quels facteurs biologiques de risque thrombotique rechercher et quelles conséquences en pratique ?

All biological risk factors that have been previously identified to increase the risk of thrombosis in adults, have also been studied in neonates with arterial Ischemic Stroke (NAIS), but most studies were retrospective and included relatively low numbers of affected children. We therefore could not suggest recommendations with a strong level of evidence and only expert proposals potentially useful for clinical practice will be presented in this text. Despite these limitations, the extensive analysis of published data supported that factor V Leiden (FVL) and increased levels of Lp(a) could be significant risk factors for NAIS. Importantly, these 2 risk factors cannot be considered as having provoked NAIS, and moreover, they do not influence the prognosis and the immediate treatment. However, since the FVL may have an impact for the prescription of a thromboprophylaxis when the neonate will become adult, to look for its presence in affected patients may be justified. For clinical practice, the following propositions can be applied: 1. Routine testing for thrombophilia (AT, PC PS deficiency, FV Leiden or FII20210A) or for detecting other biological risk factors such as antiphospholipid antibodies, high FVIII, homocystein or Lp(a) levels, MTHFR thermolabile variant, should not be considered in neonates with NAIS. 2. Testing for FV Leiden can be performed in case of documented family history of venous thromboembolic disease. 3. Testing neonates for the presence of antiphospholipid antibodies (APA) is mandatory in case of clinical events suggesting antiphospholipid syndrome in the mother (vascular thrombosis, and/or pregnancy morbidity). 4. Routine testing for thrombophilia is not proposed in both parents in case of early death of the neonate, apart from APA in the mother.

5B9, a monoclonal antiplatelet factor 4/heparin IgG with a human Fc fragment that mimics heparin-induced thrombocytopenia antibodies.

Essentials No humanized monoclonal antibody was available to study heparin-induced thrombocytopenia (HIT). We developed the first anti-platelet factor 4 (PF4)/heparin antibody with a human Fc fragment. This antibody (5B9) fully mimics the effects of human HIT antibodies. 5B9 binds two regions within PF4 that may be critical for the pathogenicity of HIT antibodies. SUMMARY: Background The diagnosis of heparin-induced thrombocytopenia (HIT) is based on clinical and biological criteria, but a standard is lacking for laboratory assays. Moreover, no humanized HIT antibody is available for pathophysiological studies. Objective To characterise 5B9, a chimeric monoclonal antibody, which fully mimics the effects of human HIT antibodies. Methods/Results 5B9, a chimeric anti-platelet factor 4/heparin complexes IgG1 antibody, was obtained after immunizing specific transgenic mice. 5B9 induced heparin FcγRIIA-dependent platelet aggregation and tissue factor mRNA synthesis in monocytes. It also induced significant thrombocytopenia and thrombin generation in mice expressing human PF4 and FcγRIIA receptors. The binding of 5B9 to PF4/H complexes was inhibited by 15 of 25 HIT plasma samples and only three of 25 samples containing non-pathogenic anti-PF4/H antibodies. KKO, a murine IgG2b HIT antibody, also inhibited the binding of 5B9 to PF4/H, suggesting that epitopes recognized by both antibodies are close. A docking analysis based on VH and VL sequences of 5B9 showed that binding of 5B9 Fab to PF4 involved 12 and 12 residues in B and D monomers, respectively, including seven previously identified as critical to the formation of a PF4/KKO complex. Two regions (Asp-7 to Thr-15 and Ala-32 to Thr-38) therefore appeared important for the binding of 5B9 and KKO on PF4 modified by heparin. Conclusions 5B9 is the first anti-PF4/H monoclonal antibody with a human Fc fragment, which induces similar cellular activation as HIT antibodies. Moreover, 5B9 binds epitopes within PF4 that are likely to be critical for the pathogenicity of HIT antibodies.

[Neonatal arterial ischemic stroke: Review of the current guidelines]. / Accidents vasculaires cérébraux ischémiques artériels néonatals : synthèse des recommandations.

Neonatal arterial ischemic stroke (NAIS) is a rare event that occurs in approximately one in 5000 term or close-to-term infants. Most affected infants will present with seizures. Although a well-recognized clinical entity, many questions remain regarding diagnosis, risk factors, treatment, and follow-up modalities. In the absence of a known pathophysiological mechanism and lack of evidence-based guidelines, only supportive care is currently provided. To address these issues, a French national committee set up by the French Neonatal Society (Société française de néonatologie) and the national referral center (Centre national de référence) for arterial ischemic stroke in children drew up guidelines based on an HAS (Haute Autorité de santé [HAS]; French national authority for health) methodology. The main findings and recommendations established by the study group are: (1) among the risk factors, male sex, primiparity, caesarean section, perinatal hypoxia, and fetal/neonatal infection (mainly bacterial meningitis) seem to be the most frequent. As for guidelines, the study group recommends the following: (1) the transfer of neonates with suspected NAIS to a neonatal intensive care unit with available equipment to establish a reliable diagnosis with MRI imaging and neurophysiological monitoring, preferably by continuous video EEG; (2) acute treatment of suspected infection or other life-threatening processes should be addressed immediately by the primary medical team. Persistent seizures should be treated with a loading dose of phenobarbital 20mg/kg i.v.; (3) MRI of the brain is considered optimal for the diagnosis of NAIS. Diffusion-weighted imaging with apparent diffusion coefficient is considered the most sensitive measure for identifying infarct in the neonatal brain. The location and extent of the lesions are best assessed between 2 and 4 days after the onset of stroke; (4) routine testing for thrombophilia (AT, PC PS deficiency, FV Leiden or FII20210A) or for detecting other biological risk factors such as antiphospholipid antibodies, high FVIII, homocysteinemia, the Lp(a) test, the MTHFR thermolabile variant should not be considered in neonates with NAIS. Testing for FV Leiden can be performed only in case of a documented family history of venous thromboembolic disease. Testing neonates for the presence of antiphospholipid antibodies should be considered only in case of clinical events arguing in favor of antiphospholipid syndrome in the mother; (5) unlike childhood arterial ischemic stroke, NAIS has a low 5-year recurrence rate (approximately 1 %), except in those children with congenital heart disease or multiple genetic thrombophilia. Therefore, initiation of anticoagulation or antithrombotic agents, including heparin products, is not recommended in the newborn without identifiable risk factors; (6) the study group recommends that in case of delayed motor milestones or early handedness, multidisciplinary rehabilitation is recommended as early as possible. Newborns should have physical therapy evaluation and ongoing outpatient follow-up. Given the risk of later-onset cognitive, language, and behavioral disabilities, neuropsychological testing in preschool and at school age is highly recommended.

Improved PCR method for amplification of GC-rich DNA sequences.

Most housekeeping genes, tumor-suppressor genes, and approx 40% of tissue-specific genes contain G+C sequences in their promoter region that were very difficult to amplify. In this report, we propose an improved polymerase chain reaction (PCR) method to be used for successful amplification of the tissue factor pathway inhibitor (TFPI)-2 gene promoter region that exhibit >70% G+C content in a sequence of approx 300 bp and a complete CpG island region spanning exon 1, the three transcription initiation sites, and the translation start site. Therefore, this method can be recommended to amplify other GC-rich genomic templates.

Repertoire cloning of a human IgG inhibitor of alpha IIB beta 3 function. The OG idiotype.

A patient (OG) with Glanzmann thrombasthenia became refractory to platelet transfusion following the production of IgG antibodies (Ab1) specific for the integrin subunit beta 3. We generated recombinant VH and VL cDNA libraries using IgG-specific mRNA isolated from OG peripheral blood B-lymphocytes that had been selected for binding to antigen (alpha IIb beta 3 adsorbed to plastic dishes). These antigen-specific B-lymphocytes contain rearranged VH DNA segments that belong exclusively to the VH4 gene family. Recombinant Fab were expressed on the surface of filamentous phage coinfected with VH and VL segments cloned into the phagemid pHEN1 or the phage fd-tet-DOG1. To facilitate selection of the desired recombinant Ab1 Fab, we developed a rabbit polyclonal antibody specific for affinity-purified OG anti-beta 3 Fab (Ab2). Ab2 reacts specifically with Ab1, and this interaction is inhibited by purified alpha IIb beta 3. Following three rounds of phage selection on Ab2 adsorbed to plastic dishes and random reassociation of heavy and light chains, we isolated Ab1 Fab and tested their binding to alpha IIb beta 3. Five Id-positive Fab were selected for further characterization. These Fab use one of two VH genes (H21 or H23) complexed with one of three V lambda genes. Subsequent sequence data demonstrated that all three lambda genes are the same clone L22 which uses a germline V lambda gene segment. Fab using H23 bind to alpha IIb beta 3, while those using H21 do not. Based on sequence homology, both H21 and H23 use VH gene segments belonging to the VH4 gene family. Thus, the idiotype OG is restricted to the VH4 gene family and is the first sequenced prototype of human antibodies that bind close to or at a functional epitope(s) of alpha IIb beta 3.

Anti-A2 and anti-A1 domain antibodies are potential predictors of immune tolerance induction outcome in children with hemophilia A.

BACKGROUND: Hemophilia A (HA) is a congenital bleeding disorder resulting from factor VIII deficiency. The most serious complication of HA management is the appearance of inhibitory antibodies (Abs) against injected FVIII concentrates. To eradicate inhibitors, immune tolerance induction (ITI) is usually attempted, but it fails in up to 30% of cases. Currently, no undisputed predictive marker of ITI outcome is available to facilitate the clinical decision. OBJECTIVES: To identify predictive markers of ITI efficacy. METHODS: The isotypic and epitopic repertoires of inhibitory Abs were analyzed in plasma samples collected before ITI initiation from 15 children with severe HA and high-titer inhibitors, and their levels were compared in the two outcome groups (ITI success [n = 7] and ITI failure [n = 8]). The predictive value of these candidate biomarkers and of the currently used indicators (inhibitor titer and age at ITI initiation, highest inhibitor titer before ITI, and interval between inhibitor diagnosis and ITI initiation) was then compared by statistical analysis (Wilcoxon test and receiver receiver operating characteristic [ROC] curve analysis). RESULTS: Whereas current indicators seemed to fail in discriminating patients in the two outcome groups (ITI success or failure), anti-A1 and anti-A2 Ab levels before ITI initiation appeared to be good potential predictive markers of ITI outcome (P < 0.018). ROC analysis showed that anti-A1 and anti-A2 Abs were the best at discriminating between outcome groups (area under the ROC curve of > 0.875). CONCLUSION: Anti-A1 and anti-A2 Abs could represent new promising tools for the development of ITI outcome prediction tests for children with severe HA.

ELISA for the detection of anticardiolipin antibodies. High specificity based on the use of adult bovine serum as buffer and systematic subtraction of non-specific binding.

We have used an ELISA procedure to compare the reactivity of samples with or without IgG anticardiolipin antibodies (ACA) on cardiolipin-coated wells (target wells) and cardiolipin-free wells (control wells), using 10% fetal calf serum (10% FCS), 10% adult bovine serum (10% ABS) or 1% bovine serum albumin (1% BSA) as buffer. With 1% BSA, ACA reactivity was very low which suggests that this buffer would be inappropriate for use in ELISA procedures for ACA. 10% FCS induced non-specific binding of normal IgG only on target wells, particularly in the case of IgG hypergammaglobulinemia. With 10% ABS, this non-specific binding occurred on the solid phase with and without cardiolipin and could be accurately evaluated by subtracting the absorbance of control wells. Results of assays on 35 systemic lupus erythematosus sera using 10% ABS as buffer showed that highly specific results-could be obtained only if non-specific binding was systematically subtracted.