Implementation of a hepatitis A prevention policy in haemophiliacs: results from the French cohort.

In the early nineties, the occurrence of hepatitis A outbreaks in some patients with haemophilia in some countries led French health authorities to recommend hepatitis A virus (HAV) vaccination in HAV-seronegative haemophiliacs. The French 'Suivi thérapeutique National des Hémophiles' cohort permitted to assess the implementation of this recommendation by the analysis of the vaccinal process, i.e. HAV seropositivity assessment and vaccination of HAV-seronegative patients, in a survival approach. In a subgroup of 812 patients diagnosed earlier than 1990 (prevalent cohort), the implementation of vaccinal process increased quickly from 0% in 1993 to 41.8% in 1994 and to 71.2% in 1996, suggesting a 'notification effect'. The vaccinal process was associated to three cofactors in a Cox model analysis (age, severity of haemophilia, centre of treatment). No infection was observed during the survey in this group. In another subgroup of 201 boys born since 1993 (incident cohort), 27.5% and 15.4% patients remained exposed to the risk at 3 and 5 years from diagnosis respectively, again with a 'centre effect', which might be linked to various factors such as regain in confidence for products or economic reasons. Only five infectious seroconversions were assessed over the 7-year survey, which represents 14.5 cases per 1000 person-year incidence without any relationship with products. Our data combined with the contemporary hepatitis A epidemiology and the current safety of anti-haemophilic concentrates, should lead to a new assessment of the risk of hepatitis A in haemophiliacs. We suggest that among patients with bleeding disorder, as well as in other populations, HAV prevention policy might be stressed on those who already suffer from chronic liver disease and/or travel in endemic countries.

[Willebrand factor deficiency and septorhinoplasty]. / Déficit en facteur Willebrand et septorhinoplastie.

INTRODUCTION: Willebrand disease can be diagnosed late, sometimes only when hemorrhage complicates surgery. French guidelines do not recommend investigation before surgery when no personal or familial hemorrhagic diathesis is reported. OBJECTIVE: To consider the advantages of Willebrand factor dosage before septorhinoplasty. METHOD: Three cases of septorhinoplasty and Willebrand factor deficiency complicated with hemorrhage compromising the functional result are reported. The routine tests (platelet count, bleeding time, and activated partial thromboplastin time) and Willebrand factor dosage were done before or after surgery. RESULTS: In the three cases, no personal or familiar hemorrhagic diathesis was found. For two cases, a hemorrhage occurred during surgery. One of them had prolonged and repeated nose bleedings after surgery. In this case, iterative packings damaged the result of surgery and a new rhinoplasty had to be done. In one case, a prolonged activated partial thromboplastin time before surgery revealed a Willebrand factor deficiency, leading to prophylactic treatment (desmopressin) of bleeding. CONCLUSION: The cases described suggest that systematic dosage of Willebrand factor before septorhinoplasty could be advantageous and that functional prognosis can be impaired by uncontrolled epistaxis.

Intracranial haemorrhages in French haemophilia patients (1991-2001): clinical presentation, management and prognosis factors for death.

Intracranial haemorrhage (ICH) is known to be a severe although uncommon complication of haemophilia. A national survey has been conducted in France in order to collect information about ICHs which occurred in haemophiliacs between 1991 and 2001 and to propose recommendations for the diagnostic and treatment of ICH. Within this period, 123 episodes of ICH were recorded from 106 patients. Two-thirds of ICH concerned patients with severe haemophilia. Half of the cases occurred in patients under 15 years of age, 67.2% of which were post-traumatic. Ten cases occurred in neonates with three fatal outcomes. Overall mortality was high (21.9%) suggesting that availability of clotting factor concentrates has not improved the prognosis of this event. Morbidity was also high with 60% of long-term sequelae. The following parameters have been identified as prognostic factors for death: thrombocytopenia, HCV infection, intraventricular or intraparenchymatous haemorrhage. A delay in diagnosis was mentioned in 43.3% of cases, often related to the lack of recognition of the initial symptoms, which may be very common (apathy, tearfulness in young children and headache in elder patients). Delayed replacement therapy was recorded in 37.2% of cases. Emergency units initially dealt with half of these patients. Information concerning recognition and management of these episodes, not only in severe haemophilia, but also in moderate and mild forms, should be regularly supplied to paediatricians in maternity and physicians from emergency units, as well as to patients and their relatives.

Analysis of biological phenotypes from 42 patients with inherited factor VII deficiency: can biological tests predict the bleeding risk?

BACKGROUND AND OBJECTIVES: Inherited factor VII (FVII) deficiency is a rare bleeding disorder characterized by a poor relationship between reported FVII clotting activity (FVII:C) and bleeding tendency. Our study was aimed at defining biological parameters that are possibly predictive for bleeding risk in this condition. DESIGN AND METHODS: Forty-two FVII-deficient patients (FVII:C <30%) were classified into two opposite clinical groups defined as severe and non-or-mild bleeders. For each patient, plasma samples were collected and then investigated for FVII:C (using a sensitive method and human recombinant thromboplastin as the reagent), FVII antigen, activated FVII coagulant activity (FVIIa:C) and the free-form of tissue factor pathway inhibitor. RESULTS: None of these tests could be used as highly accurate predictors of bleeding. Nevertheless, both FVII:C and FVIIa:C differed significantly between the two clinical groups. Using ROC-curve analysis, two critical values of 8% and 3mIU/mL for FVII:C and FVIIa:C, respectively, could be proposed to discriminate between severe bleeders and non-or-mild bleeders. INTERPRETATION AND CONCLUSIONS: A highly accurate diagnostic test for predicting bleeding tendency in inherited FVII deficiency still eludes definition, highlighting the fact that factors other than FVII itself interfere with the expression of bleeding phenotypes in this condition. Nevertheless, potential critical values using sensitive FVII:C and FVIIa:C methods may be useful in clinical laboratories for FVII-deficient patients. Those patients with FVII:C levels higher than 8% FVII:C or FVIIa:C higher than 3 mIU/mL, with no other hemostatic defect, seem to have a minimal risk of severe bleeding. Extended clinical studies are needed to support these findings.

Functional mapping of anti-factor IX inhibitors developed in patients with severe hemophilia B.

Development of inhibitory antibodies is a serious complication of treatment with repeated factor IX infusions in a minority of patients with hemophilia B. Such antibodies detected in 8 patients have been characterized. Typing studies revealed that patients' immune response toward factor IX is highly heterogeneous and involves immunoglobulin G (IgG) antibodies, preferentially IgG1 and IgG4. The preservation of the sequence and the 3-dimensional orientation of the amino acids constituting one epitope are highly important for the assembly of an antibody-antigen complex. To localize the epitopes on the factor IX molecule, an original approach was designed using a set of factor X chimeras carrying regions of factor IX. Results showed that some patients' antibodies were directed against both the domain containing the gamma-carboxy glutamic acid residues (Gla domain) and the protease domain of factor IX. In contrast, no binding was observed to the epidermal growth factor-like domains or to the activation peptide. Functional characterization showed that the purified IgG from patients' serum inhibited the factor VIIIa-dependent activation of factor X. Moreover, patients' IgG directed against the Gla domain inhibited the binding of factor IX to phospholipids as well as the binding of factor VIII light chain to factor IXa. These data demonstrate that inhibitors appearing in patients with severe hemophilia B display specificity against restricted functional domains of factor IX.

Interferon alpha therapy in haemophilic patients with chronic hepatitis C: a French multicentre pilot study of 58 patients.

BACKGROUND AND OBJECTIVES: Information about the long-term efficacy of interferon alpha (interferon-alpha) in haemophilic patients with chronic hepatitis not co-infected with the human immunodeficiency virus (HIV-1) is still limited. Previous studies seemed to indicate a low rate of response. The aim of this study was to evaluate the safety and long-term efficacy of interferon treatment in multi-transfused haemophiliacs. METHODS: Fifty-eight haemophiliacs were scheduled to receive 3 MU of interferon-alpha 2b three times a week for 12 months. The patients were followed up for at least 24 months post-treatment. Response was assessed by measurements of serum hepatitis C virus (HCV) RNA. RESULTS: Twenty-four patients (41.4%) dropped out. Except for seven patients, the symptoms that led to interrupting interferon treatment would probably not have resulted in the same decision in non-haemophilic patients. One patient developed an inhibitor to the deficient clotting factor without haemorrhagic consequences. In an intent to treat, the sustained virological response rate was 14%. However, when considering only the 34 patients who received the full treatment, HCV-RNA was cleared in eight patients (23%). CONCLUSIONS: This study suggests that multi-transfused haemophiliacs with chronic hepatitis not co-infected with HIV-1 respond to prolonged treatment with interferon-alpha in a similar proportion to that observed in non-haemophiliacs. There was a high rate of patients who did not complete the interferon-alpha treatment, and this seems to be characteristic of this patient population.

Role of the leucine-rich domain of platelet GPIbalpha in correct post-translational processing--the Nancy I Bernard-Soulier mutation expressed on CHO cells.

The mechanisms governing the biosynthesis and surface expression of platelet adhesive receptors on parent megakaryocytes are as yet poorly understood. In particular, the assembly and processing of the multisubunit glycoprotein (GP) Ib-IX-V complex, a receptor for von Willebrand factor (vWf) is not fully understood. In the present work, these questions were addressed by reproducing a natural mutation of GPIbalpha found in a variant case of Bernard-Soulier syndrome (Nancy I), due to the deletion of leucine 179 in the seventh leucine-rich repeat of the polypeptide. Wild type and mutated GPIbalpha were transfected into CHO cells expressing GPlbbeta and GPIX. Flow cytometry showed surface expression of the three subunits of both GPIb-IX complexes, but GPlbalphadeltaLeu was present at lower levels (20-40%) and was recognized only by a sub class of monoclonal antibodies which epitopes were not modified by the mutation. These properties reproduce the defect found in the patient's platelets, demonstrating the causative nature of the mutation and validate the use of the CHO cells model. Biochemical studies were performed in an attempt to elucidate the mechanism of the conformational change of GPIbalphadeltaLeu. They unexpectedly revealed a major glycosylation deficiency of the mutated GPIbalpha leading to a 40% decrease in molecular weight. The other two subunits of the complex were however normal and present at the plasma membrane. The deletion led to complete functional deficiency with lack of vWf binding of CHOalphadeltaLeu transfected cells in the presence of botrocetin and defective adhesion to a vWf coated surface under static conditions. Finally, in contrast to normal CHOalphabetaIX cells, which displayed rolling and deceleration when perfused over a vWf surface, CHOalphadeltaLeubetaIX cells were unable to roll over or attach to a vWf substratum. These results show that the integrity of the leucine-rich region of GPIbalpha is essential for normal processing and function of the GPIb-IX complex. In addition, these results obtained in a cellular system supported the suspected role of the macroglycopeptide region of GPIbalpha in maintaining a suitable conformation of this multisubunit receptor to perform its adhesive function.

[Heparin-induced thrombopenia]. / Thrombopénie induite par l'héparine.

Heparin-induced thrombocytopenia remains a topical subject for at least two reasons. The first reason is the increasing prescription of low molecular weight heparins (LMWH) rather than unfractionated heparins, with limited laboratory surveillance, raising the question concerning the need for twice-weekly platelet counts, according to the recommendations of the Vidal drug directory. The second reason is the recent release onto the market of two products, danaparoid (Orgaran) and lepirudin (Refludin) for this precise indication of heparin-induced thrombocytopenia. These products greatly facilitate the management of this complication. Many basic research teams are trying to optimize the detection of heparin-dependent antibodies and to more clearly elucidate the mechanism of this particular thrombocytopenia, which carries a risk of very severe thrombotic complications when the diagnosis is delayed.

A cross-over pharmacokinetic study of a double viral inactivated factor IX concentrate (15 nm filtration and SD) compared to a SD factor IX concentrate.

A double blind randomized cross-over multi-center study has been conducted to compare the pharmacokinetic and coagulation activation markers of high-purity factor IX concentrate subjected to both solvent/ detergent (SD) treatment and 15 nm-filtration (FIX-SD-15) with the licensed product subjected only to solvent-detergent (FIX-SD). This filtration process allows the elimination of small particles, such as non-enveloped viruses (i.e., hepatitis A and parvovirus B19). Eleven severe hemophilia B patients (FIX coagulant activity <2 IU/dl) received one infusion of 60 IU/kg of FIX-SD and one infusion of 60 IU/kg of FIX-SD-15 at least at 10 days interval. Blood samples were obtained before and at various time up to 72 h after infusion. The decay curves of factor IX (FIX:C and FIX:Ag) were evaluated by a model independent method. Bioequivalence was found between the two concentrates using the Schuirmann test. The mean FIX:C and FIX:Ag recovery of FIX-SD-15 was 1.08 and 0.89 IU/dl/IU/kg respectively with a mean half-life of 33.3 h for FIX:C and 25.6 h for FIX:Ag. Six months after initial enrollment, pharmacokinetic parameters were similar in the 7 patients tested. There was no significant variation of prothrombin fragment 1+2 and thrombin-antithrombin complexes measured up to 6 h after infusion, indicating that there was no activation process after administration of FIX. In conclusion, these data demonstrate that the introduction of a 15 nm filtration does not alter the pharmacokinetic profile of a well characterized SD FIX concentrate while providing additional viral safety.

Experiences with continuous infusion of recombinant activated factor VII.

A questionnaire was sent to 28 haemophilia treatment centres known to have used recombinant activated factor VII (rFVIIa; NovoSeven, Novo Nordisk, Bagsvaerd, Denmark), to collect data on continuous infusion of this product. This mode of administration was recently introduced for rFVIIa but there are several questions which remain to be answered in order to optimize this technique. Of the 26 responding centres, 14 had used rFVIIa in continuous infusion for 40 treatment episodes over a total of 283 days. In most of the cases the treatment was targeted at a factor VII level of 10 IU/ml, monitored by the one-stage clotting assay. This seemed to be adequate for most of the haemorrhagic and surgical procedures. Pretreatment pharmacokinetic evaluation was performed in only a minority of the cases but is probably of great importance given the wide variation observed in the clearance values. A strategy was necessary to prevent local thrombophlebitis, at least for infusions in peripheral veins; parallel infusion of heparin, saline or dextrose-saline proved effective. The question of optimal monitoring needs further attention. Haemorrhagic complications were significantly less frequent when treatment was combined with the antifibrinolytic tranexamic acid.

[Role of the hemostasis laboratory in the etiologic approach to deep vein thrombosis]. / Place du laboratoire d'hémostase dans la démarche étiologique des thromboses veineuses profondes.

Thrombophilia is characterized by an inherited or acquired defect in the blood coagulation pathway leading to an increased risk for thrombosis. The etiological approach following confirmed venous thrombotic events should rule out medical or chirurgical risk factors. Thrombophilia should be sought by laboratory tests. The recent discovery of a blood coagulation defect: inherited resistance to activated protein C which is found to 20% of patients with former thrombotic events has changed current laboratory approach. Deficiencies of one of the anticoagulant proteins (antithrombin III, protein C, protein S) are found in 10% of the patients, similar to the frequency of antiphospholipid antibodies. These tests may be difficult to interpret immediately after the thrombotic event because of various factors such as inflammatory states or anticoagulant treatments. Therefore this abnormal tests should be confirmed on a later sample analysis far from the event. The discovery of an inherited blood coagulation pathway defect may affect the duration of treatment, prophylaxis in situations with circumstantial risk factors and requires familial analysis. Inherited resistance to activated protein C may be associated with another inherited defect leading to an increased risk for thrombosis.

Comparative assessment of phospholipid-binding antibodies indicates limited overlapping.

The implication of autoantibodies with anticoagulant and/or so-called antiphospholipid activities, under clinical circumstances with vascular obliteration, has led to the development of various types of tests allowing their detection. The most used tests involve investigation of the presence of an anticoagulant effect and of anticardiolipin IgG. It has also been proposed that the reactivity of patient samples toward other phospholipids or proteins be tested, but it remains difficult to appreciate which tests are redundant or complementary. Here we investigated whether the dissociation or association of anticoagulant and anticardiolipin correlated with specific ELISA reactivity to five other phospholipids: phosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylcholine, and phosphatidylethanolamine. The study was performed with 70 samples, evenly partitioned as positive for either anticardiolipin antibodies or anticoagulant effect, or both. Our data clearly confirm that cardiolipin reactivity is an individual entity, likely to be complementary to other assays. Neither anticardiolipin nor anticoagulant levels correlated with assays investigating antibody levels toward the five other phospholipids, although higher mean levels were noted when both lupus anticoagulant and anticardiolipin antibodies are present. Individual patterns were evidenced in all groups. These data support the interest of current and further studies exploring the clinical relevance of individual reactivities to phospholipids.

A three-base deletion removing a leucine residue in a leucine-rich repeat of platelet glycoprotein Ib alpha associated with a variant of Bernard-Soulier syndrome (Nancy I).

Leucine-rich repeats are conserved structural motifs present in the four components of the human platelet glycoprotein Ib/IX/V complex receptor for the adhesive protein von Willebrand factor. The absence or abnormality of this complex is responsible for Bernard-Soulier disease, an autosomal recessive bleeding disorder. We report a deletion of leucine 179, located in a highly conserved position of the seventh leucine-rich repeat of GPIb alpha, found in a variant form of Bernard-Soulier disease (Bernard-Soulier Nancy I). Three affected siblings of a family were characterized by absence of ristocetin-induced platelet agglutination, although ADP aggregation was normal. Flow cytometry studies showed detectable amounts of all four members of the GPIb/IX/V complex on the surface of the patients' platelets. Western blotting revealed normal levels of GPIX, decreased levels of GPIb beta and GPV, and < 1% of GPIb alpha. RT-PCR studies showed the presence of mRNA coding for GPIb alpha, GPIb beta, GPIX and GPV. Sequencing showed a three-base deletion which results in the absence of a leucine residue, highly conserved across the seven leucine-rich repeats of GPIb alpha and also within the other members of the leucine-rich glycoprotein family. The absence of the leucine 179 in a patient's GPIb alpha is believed to cause a conformational change in the protein which would account for the lack of binding of most of the MoAbs tested and would be responsible for the absence of von Willebrand factor binding. These results point to the leucine-rich region of GPIb alpha as being required for the correct exposure of the von Willebrand binding site as well as for the correct assembly and stability of the GPIb/IX/V complex on the platelet surface.

Hemorheological and hemostatic parameters in children with nephrotic syndrome undergoing steroid therapy.

Disturbances in hemostatic and hemorheological parameters have been investigated in a group of 29 children with nephrotic syndrome: 23 children classified as steroid-sensitive and 6 as steroid-resistant. Studies were performed before prednisone treatment and 3 weeks later, after initiation of steroid therapy. Before treatment, the alterations in hemostatic system involved moderate thrombocytosis with spontaneous aggregation in 19 patients. High levels of fibrinogen, factor VIII, Willebrand factor, protein C, protein S and alpha 2-macroglobulin (alpha 2M) were observed. Factor XII and alpha 1-antitrypsin (alpha 1AT) were lower than normal. Antithrombin III (ATIII) level was normal in the majority of patients. A plasma and blood hyperviscosity syndrome was also observed as well as an increase in erythrocyte aggregation. During treatment, an improvement in the hemostatic parameters was observed in the patients who responded to prednisone. The expected increase in factor VIII (frequently described in the literature) was not observed, while there was a significant increase in protein C. In the steroid-resistant patients, the only significant changes observed were decreased fibrinogen and increased protein C. The hemorheological parameters showed a tendency towards normality regardless of whether or not the treatment provided remission of NS. The relationship between hemorheological and hemostatic factors changes are discussed.

[Development of antifactor VIII circulating anticoagulant after surgery of the biliary tract]. / Apparition d'un anticoagulant circulant antifacteur VIII après chirurgie de la voie biliare.

In a 25-year-old woman, admitted with a haemorrhagic syndrome following biliary surgery, an inhibitor of factor VIII was detected. As bleeding was major, she was re-operated on under perioperative administration of the anti-inhibitory coagulant complex Autoplex-T, associated with polyvalent i.v. immunoglobulins. The other therapeutic agents are also considered and their indications discussed, after a review of the circumstances of the diagnosis of this disease.

Hepatitis A in French hemophiliacs.

The prevalence of serum antibodies to hepatitis A virus (HAV) in 793 hemophilia A (HA) and 89 hemophilia B (HB) patients coming from 10 French Hemophilia Centers and treated since 1986-1987 with solvent/detergent (SD)-treated products is reported. The results indicated seropositivity to HAV of 29.9% in HA and 40.4% in HB patients. There was no difference among the patients according to severity of the disease, HIV serology or administration of factor VIII during the last 12 months. Seropositivity increased with age from 5.2% in HA children to 42.4% in adults (in HB the respective prevalences in the same groups were 7.7% and 56.1%). When compared to normal controls (n = 585), the prevalence of HAV seropositivity was not excessive in HA patients (n = 206). 19/20 children exclusively treated with a very-high-purity SD-factor VIII concentrate (Centre régional de transfusion sanguine, Lille) remained HAV seronegative. Six cases of HAV contamination were reported in patients with severe HA, probably reflecting the level of HAV endemy in the normal population in France. No special risk of HAV transmission linked to the SD products used in France since 1986 had thus been identified.

Interlaboratory evaluation of methods for the assay of Protein C in purified concentrates.

Determination of the quantity and activity of the Protein C molecule is of the utmost importance in highly purified concentrates prepared for replacement therapy. A multicenter study was undertaken to evaluate the comparability and accuracy of Protein C assays from commercial sources. Significant between-assay and interlaboratory differences were found for both functional and immunological assays. The interlaboratory variability is explained in part by the use of different control plasmas. The results also indicate the importance of the diluent used. This study emphasizes the need for standardized methods for determining the characteristics of Protein C concentrates.