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® .

Two novel variants of uncertain significance in GP9 associated with Bernard-Soulier syndrome: Are they true mutations?

Bernard-Soulier syndrome (BSS) is an autosomal recessive major thrombocytopathy, the symptoms of which are mainly marked by mucocutaneous bleeding. This rare disease, initially described in the 1970s, is the result of an abnormal formation of the glycoprotein complex Ib-IX-V (GP Ib-IX-V), a platelet receptor of von Willebrand factor. A large number of mutations, sometimes involving the GP9 gene, have been described as possibly responsible for the disease. We report here the case of a BSS patient who presented with persistent thrombocytopenia (31x109/L) and decreased surface expression of GPIb-IX-V on large platelets with anisocytosis. Thorough molecular analyses disclosed two previously unreported GP9 variants, respectively c.230T>A (p.Leu77Gln) and c.255C>A (p.Asn85Lys). Both are likely to modify the conformation of GP-IX interactions with other glycoproteins of the Ib-IX-V complex and thus proper expression of this complex on the membrane of platelets.

Platelet function analyser (PFA-100) results and von Willebrand factor deficiency: a 16-year 'real-world' experience.

The platelet function analyser (PFA-100) is a biological tool designed to explore primary haemostasis. This system has thus been widely demonstrated as reliable in detecting von Willebrand factor (VWF) deficiency. However, most studies were based on patients benefitting from regular medical care and accurate diagnosis, and it would seem probable that the results were somewhat optimistic, and do not reflect its performances in 'real-world' situations. We have chosen to study the reliability of PFA-100 for screening VWF ristocetin cofactor (VWF:RCo) deficiency. We retrospectively analysed the results (n = 6431) of 4027 patients referred to our centre between October 1997 and June 2013 and in whom PFA-Epi, PFA-ADP, and VWF:RCo activity had been evaluated. We studied the influence of blood group on the results and the performances of each method in a subgroup of 213 patients with genetically confirmed von Willebrand disease. We have shown that the PFA-100 system, in our experience, constitutes an excellent screening test for detecting VWF:RCo deficiency, whatever the clinical situation, in 'real-world' conditions. The negative predictive value (NPV), the positive predictive value, the sensitivity and the specificity were respectively: 0.98, 0.51, 0.98 and 0.40. When values adjusted for blood group are used, NPV and sensitivity are inferior to those using normal values which have not been adjusted for blood group. We have shown the PFA-100 method to be more efficient in screening for VWF deficiency than the VWF:RCo technique.

Diagnosis and management challenges in patients with mild haemophilia A and discrepant FVIII measurements.

Thirty per cent of patients with mild haemophilia A (MHA) present markedly different FVIII: C level when assayed by one-stage clotting and two-stage chromogenic assays. It is, therefore, a real clinical challenge to predict the individual bleeding risk of these patients. The aim of the present work was to study the relationship between the bleeding tendency of these patients with the results of a panel of phenotypic and genotypic tools. Thirty-six patients with MHA were included in this multicentre prospective clinical study. The severity of bleeding symptoms was evaluated using the ISTH/SSC score. FVIII:C levels were measured using an activated partial thromboplastin time-based one-stage FVIII assay (FVIII: C1) and three commercial chromogenic kits (FVIII:CR). FVIII antigen levels, thrombin generation measurement and FVIII gene mutation analysis were also performed. Our results showed that a one-stage FVIII: C assay cannot rule out the diagnosis of MHA, a combined use of FVIII:C1 with a FVIII:CR is suitable for detecting MHA. We observed that FVIII:CR results better reflected the clinical bleeding tendency of patients compared to FVIII:C1. We also observed a relationship between thrombin generation (TG) capacity and FVIII:CR of these patients. FVIII gene mutation analysis showed mutations previously reported in MHA patients with discrepant FVIII:C measurements, but with no predictive value of the individual bleeding phenotype of patients. Overall, we observed a relationship between chromogenic FVIII:C results, TG assay and bleeding tendency of patients with discrepant FVIII:C measurements, while FVIII:C1 was not well correlated with clinical bleeding phenotype in this particular population.

Management of bleeding in severe factor V deficiency with a factor V inhibitor.

Factor V (FV) inhibitor arises rarely after using fresh frozen plasma (FFP) to treat inherited FV deficiency and is often a real therapeutic challenge. Here, we report a patient with a severe FV deficiency who developed such an inhibitor and was then treated with recombinant activated FVII (rFVIIa) and platelet concentrates (PC). Monitoring was assessed by thrombin generation assay (TGA). PC were more effective than rFVIIa in treating bleeding, but there was no correlation between the TGA results and clinical efficacy.

Introduced Siberian chipmunks (Tamias sibiricus barberi) harbor more-diverse Borrelia burgdorferi sensu lato genospecies than native bank voles (Myodes glareolus).

Little attention has been given in scientific literature to how introduced species may act as a new host for native infectious agents and modify the epidemiology of a disease. In this study, we investigated whether an introduced species, the Siberian chipmunk (Tamias sibiricus barberi), was a potentially new reservoir host for Borrelia burgdorferi sensu lato, the causative agent of Lyme disease. First, we ascertained whether chipmunks were infected by all of the B. burgdorferi sensu lato genospecies associated with rodents and available in their source of infection, questing nymphs. Second, we determined whether the prevalence and diversity of B. burgdorferi sensu lato in chipmunks were similar to those of a native reservoir rodent, the bank vole (Myodes glareolus). Our research took place between 2006 and 2008 in a suburban French forest, where we trapped 335 chipmunks and 671 voles and collected 743 nymphs of ticks that were questing for hosts by dragging on the vegetation. We assayed for B. burgdorferi sensu lato with ear biopsy specimens taken from the rodents and in nymphs using PCR and restriction fragment length polymorphism (RFLP). Chipmunks were infected by the three Borrelia genospecies that were present in questing nymphs and that infect rodents (B. burgdorferi sensu stricto, B. afzelii, and B. garinii). In contrast, voles hosted only B. afzelii. Furthermore, chipmunks were more infected (35%) than voles (16%). These results may be explained by the higher exposure of chipmunks, because they harbor more ticks, or by their higher tolerance of other B. burgdorferi sensu lato genospecies than of B. afzelii. If chipmunks are competent reservoir hosts for B. burgdorferi sensu lato, they may spill back B. burgdorferi sensu lato to native communities and eventually may increase the risk of Lyme disease transmission to humans.

[Tailless tenrec (Tenrec ecaudatus): natural maintenance host of leptospires?]. / Le tanrec (Tenrec ecaudatus): réservoir animal de leptospires?

In order to know if the Tailless tenrec (Tenrec ecaudatus), endemic insectivorous mammal of Madagascar and present only on Indian Ocean islands, is a natural maintenance host of leptospires carrier in La Reunion, we conducted a research of anti-leptospire antibodies by microagglutination test in 37 individuals. 81.1% of serums tested were positive, (> 1/50) with the highest titers for the Icteroharmorrhagiae serogroup. So, in la Reunion, the Tailless tenrec can be suspected of being a reservoir of leptospires. A more detailed study should confirm or not this hypothesis and should possibly quantify its importance.

Molecular basis of severe factor XI deficiency in seven families from the west of France. Seven novel mutations, including an ancient Q88X mutation.

Inherited factor (F)XI deficiency is a rare disorder in the general population, though it is commonly found in individuals of Ashkenazi Jewish ancestry. In particular, two mutations--a stop mutation (type II) and a missense mutation (type III)--which are responsible for FXI deficiency, predominate. The bleeding tendency associated with plasma FXI deficiency in patients is variable, with approximately 50% of patients exhibiting excessive post-traumatic or postsurgical bleeding. In this study, we identified the molecular basis of FXI deficiency in 10 patients belonging to six unrelated families of the Nantes area in France and one family of Lebanese origin. As in Ashkenazi Jewish or in French Basque patients, we have identified a new ancient mutation in exon 4 resulting in Q88X, specific to patients from Nantes, that can result in a severely truncated polypeptide. Homozygous Q88X was found in a severely affected patient with an inhibitor to FXI and in three other unrelated families, either as homozygous, heterozygous or compound heterozygous states. Other identified mutations are two nonsense mutations in the FXI gene, in exon 7 and 15, resulting in R210X and C581X, respectively, which were identified in three families. A novel insertion in exon 3 (nucleotide 137 + G), which causes a stop codon, was characterized. Finally, sequence analysis of all 15 exons of the FXI gene revealed three missense mutations resulting in G336R and G350A (exon 10) and T575M (exon 15). Two mutations (T575M and G350A) with discrepant antigen and functional values are particularly interesting because most of the described mutations are associated with the absence of secreted protein.

Incidence of factor VIII inhibitor development in severe hemophilia A patients treated only with one brand of highly purified plasma-derived concentrate.

The incidence of factor VIII inhibitor was studied in a cohort of 56 previously untreated patients with severe hemophilia A (factor VIII below 1 U/dl). They received only one brand of highly purified factor VIII concentrate (HPSD-VIII) prepared by conventional chromatography with a solvent-detergent step for viral inactivation. Follow-up since the first infusion of HPSD-VIII was from 1 to 76 months (mean = 29) and cumulative exposure days (CED) from 1 to over 100 (median = 26). Five patients (9%) developed an inhibitor after 6 to 19 CED, only one being a high responder (2%), showing a low incidence of inhibitor compared with previous studies using high purity plasma-derived or recombinant products.

[Antiphospholipid syndrome. Proposition for management]. / Le syndrome antiphospholipides. Proposition pour une conduite à tenir.

Antiphospholipid antibodies (antiprothrombinase and anticardiolipin) carry with them for mothers the risks of repeated fetal loss and of disorders of the blood clotting mechanism both before and after delivery. All the same screening does not have to be carried out routinely but should be reserved for patients who have already lost one fetus (intrauterine death after 12 weeks of amenorrhoea) and/or venous or arterial thrombosis. The diagnosis depends on a strict methodology and strict criteria for making a positive diagnosis. The treatment of these antibodies (with corticosteroids and intravenous immunoglobulin) or the prevention of possible thrombotic complications (using platelet antiaggregation/heparin) has to be decided taking into account the level of antibodies, previous obstetric and thrombotic history and the lupus symptomatology as shown by the patients. The overall success rate of treatment is between 53 and 81%.

[Acquired Willebrand syndrome with lymphoproliferative disorders]. / Syndrome de Willebrand acquis avec désordres lymphoprolifératifs.

INTRODUCTION: Willebrand's syndrome is rarely acquired. We report four cases associated with lymphoproliferative syndromes. CASE REPORTS: We observed four patients with lymphoid hemopathies who developed acquired Willebrand's syndrome. Two patients had Waldenström's disease (kappa), one had a monoclonal gammapathy of undetermined signification (kappa immunoglobulin M) and the fourth had chronic lymphoid leukemia with mast cell infiltration of the skin. Anti-vWFRCo antibodies were evidenced in only 1 case. Chemotherapy, used in 3 cases, improved hemostasis in one patient. Intravenous immunoglobulins (1 patient) and desmopressin (2 patients) were ineffective. The pathogenic mechanisms and possible therapeutic approaches to acquired Willebrand's syndrome are discussed. DISCUSSION: Acquired Willebrand's syndrome rarely occurs in association with lymphoproliferative disorders appears to be uncommon but the frequency is probably underestimated because appropriate tests are not always performed. The diagnostic search is important however since the hemostasis disorders due to acquired Willebrand's syndrome could be corrected if appropriate etiological treatment is given.

[Acquired hemophilia and rheumatoid arthritis. Value of intravenous immunoglobulins]. / Hemophilie acquise et polyarthrite rhumatoïde. Intérêt des immunoglobulines intra-veineuses.

The authors report an exceedingly rare complication of rheumatoid arthritis, i.e. acquired hemophilia due to anti-factor VIII autoantibody production. Treatment with intravenous immune globulin ensured control of hemorrhagic manifestations by inducing a transient rise in factor VIII level.

[Acquired hemophilia A. A monocentric retrospective study of 39 patients]. / Hémophilie A acquise. Étude d'une série rétrospective monocentrique de 39 patients.

PURPOSE: Acquired haemophilia A (AHA) is a rare bleeding disorder, due to the presence of an inhibitor directed against factor VIII (FVIII). About 50% of the AHA are idiopathic, while the remaining 50% are related to an underlying disorder or condition (autoimmune diseases, malignancies, postpartum, etc.). PATIENTS AND METHODS: We report on a monocentric retrospective cohort of 39 patients with AHA. Data were collected and compared to recent published data. RESULTS: Thirty-nine patients were admitted for AHA between 1993 et 2011. Mean age at diagnosis was 71.3 years, and we noted a marked male predominance. Although the majority of patients presented a bleeding event at diagnosis (94.9%), the hemorrhagic mortality was low (2.6%). On the contrary, immunosuppressive morbidity and mortality were high in this elderly population. There was a clear correlation between initial FVIII inhibitor titer and complete remission delay. We did not identify prognostic factor for global survival. CONCLUSION: AHA is a rare but potentially fatal disorder. Rapidity of diagnosis and treatment initiation is crucial. Morbidity and mortality, particularly of infectious cause, due to immunosuppressive treatment, should lead to consider other available therapeutical options.

Prevalence, biological phenotype and genotype in moderate/mild hemophilia A with discrepancy between one-stage and chromogenic factor VIII activity.

BACKGROUND: In most laboratories, the severity of hemophilia A is assessed by the factor VIII activity (FVIII:C) one-stage assay. However, comparisons of these results with those of two-stage assays can reveal discrepancies and suggest misdiagnosis. PATIENTS/METHODS: In this monocentric study, we measured FVIII:C with two methods (one-stage chronometric and chromogenic assays) in 307 (173 families) patients with moderate/mild hemophilia A. To compare results, we used a chronometric/chromogenic ratio. Discrepancy was defined as a ratio < 0.5 or > 1.5. We studied their putative involvement at known FVIII functional sites, their interspecies conservation status, and their spatial position within the FVIII structure. RESULTS: Thirty-six patients from 17 families exhibited a discrepancy between the two assays: 12 (6.9%) families had a low ratio (< 0.5), and five (2.9%) families had a high ratio (> 1.5). Qualitative deficiency was diagnosed in about 16% of the families. Molecular studies were performed in 15 of these 17 families, resulting in each case in the identification of missense mutations, including three novel mutations. We were further able to propose a pathophysiologic explanation. CONCLUSIONS: In this monocentric study, we have demonstrated a discrepancy between FVIII:C assay results in 10% of families with moderate/mild hemophilia A. The prevalence of 'inverse' discrepancy (i.e. low chronometric/chromogenic ratio) is high as compared with previous reports. We suggest that both FVIII:C assays are recommended in patients with moderate/mild hemophilia A for a complete biological phenotype. This could also improve our knowledge of the FVIII structure-function relationships.

Mild hemophilia A with factor VIII assay discrepancy: using thrombin generation assay to assess the bleeding phenotype.

INTRODUCTION: In some patients with mild hemophilia A, there are discrepancies between 1-stage (1-st) and 2-stage (2-st) factor VIII (FVIII) clotting assays, and also chromogenic assays for FVIII activity (FVIII:C). We examined whether thrombography could provide a better evaluation of the hemostatic status of these patients. METHODS: Two families with such discrepancies and markedly contrasting clinical histories were studied. Family X had no serious bleedings, in contrast to family Y. Sixty-one moderate/mild hemophiliacs without discrepancy and 15 healthy subjects served as controls. Calibrated automated thrombography was performed with platelet-rich plasma after one freeze-thawing cycle and low tissue factor concentration. RESULTS: The chromogenic FVIII:C levels were higher (0.90 +/- 0.15 and 0.47 +/- 0.13 IU mL(-1)) than the 1-st clotting ones (0.14 +/- 0.05 and 0.10 +/- 0.05 IU mL(-1)) in family X and Y, respectively (P < 0.001). Mean endogenous thrombin potential (ETP) was 1579 +/- 359 nM min(-1) and 1060 +/- 450 for healthy controls and hemophilic controls, respectively. For members of family X, the ETP values were 1188, 1317 and 2277 nM min(-1), whereas for those of family Y they ranged from 447 to 1122 nM min(-1). Two novel missense point mutations were evidenced: p.Ile369Thr in family X and p.Phe2127Ser in family Y. In family X, we postulate that the mutation is responsible for a delayed but non-deleterious FVIII activation. CONCLUSIONS: Our results suggest that the hemostatic phenotype assessed by thrombography may be clinically relevant in moderate/mild hemophilic patients with discrepant FVIII:C results.

Characterization of different diamond-like carbon electrodes for biosensor design.

Diamond-like carbon (DLC) films are gaining big interest in electrochemistry research area. DLC electrodes made with different ratio of sp(3)/sp(2) carbon hybridization or doped with different percentages of nickel were characterized electrochemically by cyclic voltammetry and by amperometric measurements towards hydrogen peroxide. SiCAr1 and SiCNi5% were chosen as sensitive transducers for the elaboration of amperometric glucose biosensors. Immobilization of glucose oxidase was carried out by cross-linking with glutaraldehyde. Measurements were made at a fixed potential+1.0V in 40mM phosphate buffer pH 7.4. SiCAr1 seems to be more sensitive for glucose, 0.6875muA/mM, than SiCNi5%, 0.3654muA/mM. Detections limits were 20muM and 30muM, respectively. Apparent Michaelis-Menten constants were found around 3mM. Forty-eight percent and 79% of the original response for 0.5mM glucose remained after 10 days for both biosensors, respectively.

Factor VIII recovery after a single infusion of recalibrated ReFacto in 14 severe haemophilia A patients.

A recent multicentre collaborative study showed higher estimates of ReFacto potency when assayed with ReFacto Laboratory Standard(TM) (RLS) in comparison when standards consisting of full-length factor VIII (FVIII) were used. The RLS was hence recalibrated, leading to a 20% increase in the amount of ReFacto per vial without change in the labelled potency. The primary objective of this study was to determine the incremental and in vivo recovery of the recalibrated ReFacto in patients with severe haemophilia A. Fourteen male severe haemophilia A patients (FVIII < 1 IU dL(-1)) with a cumulative previous exposure days to any FVIII product >150 were administered an intravenous infusion 50 +/- 5 IU kg(-1) of ReFacto over a 5-min period. Blood samples were collected before infusion and after 15, 30 and 60 min. FVIII clotting activity (FVIII:C) was assessed in a central laboratory by the chromogenic substrate assay. After ReFacto infusion, peak FVIII:C was obtained within 15 min for 10 patients and within 30 min for the remaining four. Mean FVIII:C at peak was 117.7 +/- 17.3 IU dL(-1). Mean incremental recovery was 2.22 +/- 0.27 IU dL(-1) per IU kg(-1) while mean in vivo recovery was 105.9 +/- 14.6%. One patient reported three mild adverse events rated as 'unrelated' to the study drug. FVIII recovery after recalibrated ReFacto infusion falls within the expected range and is similar to the values reported for other FVIII concentrates.