Efficacy and safety during formulation switch of a pasteurized VWF/FVIII concentrate: results from an Italian prospective observational study in patients with von Willebrand disease.

Von Willebrand disease (VWD) is an inherited bleeding disorder caused by the quantitative or qualitative deficiency of von Willebrand factor (VWF). Replacement therapy with plasma-derived VWF/factor VIII (FVIII) concentrates is required in patients unresponsive to desmopressin. To assess the efficacy, safety and ease of use of a new, volume-reduced (VR) formulation of VWF/FVIII concentrate Haemate(®) P in patients requiring treatment for bleeding or prophylaxis for recurrent bleeding or for invasive procedures. Pharmacoeconomic variables were also recorded. Data were analysed using descriptive statistics. This was a multicentre, prospective, observational study. Consecutively enrolled patients received Haemate(®) P VR according to their needs, and were followed for 24 months. Of the 121 patients enrolled, 25.6% had type 3 VWD and more than 40% had severe disease. All patients were followed for 2 years, for a total of 521 visits. On-demand treatment was given to 61.9% of patients, secondary long-term prophylaxis to 25.6% and prophylaxis for surgery, dental or invasive procedures to 45.5%. The response to treatment was rated as good to excellent in >93-99% of interventions. The new formulation was well tolerated by all patients with no report of drug-related adverse events. The switch to volume-reduced Haemate(®) P was easy to perform and infusion duration was decreased twofold compared with the previous formulation. Volume-reduced Haemate(®) P was at least as effective and well-tolerated as the previous formulation.

A web-based registry of inherited bleeding disorders in the region of Emilia-Romagna: results at three and a half years.

A Registry of inherited bleeding disorders was set up in the Region of Emilia-Romagna (RER) to collect information about these diseases and to improve the quality of care. From January 2003, the eight Haemophilia Centres (HC) in the RER began to use computerized clinical records; every 6 months, they send data to Parma Hospital to be processed and published in a website (http://www.registroemofiliarer.it). Great efforts are made to ensure high quality of data. Results of general interest are included in a free 'public area' and more sensitive data in a 'reserved area' (open only to HC and to health authorities). A total of 610 individuals are included: 249 haemophilia A (HA), 63 haemophilia B (HB), 173 von Willebrand's disease, 69 rare bleeding disorders, seven platelet disorders and 49 haemophilia carriers; 131 were genotyped, 188 were tested for inhibitors (16 affected). The most frequent bleeding was haemarthrosis. The joint score (evaluated in 104 haemophiliacs) was higher in severe HA. There were 22 HIV-positive and 182 hepatitis C virus-positive patients (21% have chronic hepatitis, two hepatocellular carcinoma). In 2005, two patients received primary prophylaxis, 47 secondary prophylaxis, four children were on immune-tolerance induction. From 2003 to 2005 the use of recombinant products was greatly increased and the majority of patients received them. The mean clotting factor consumption for prophylaxis was higher than on-demand treatment. The main features of registry are to collect high quality and comprehensive data of all patients followed by HC, to improve quality of care and it's availability on the web.

A photometric method for the dosage of factor XIII applied to the study of chronic hepatopathies.

The photometric method of Fickenscher et al. for the determination of factor XIII (FXIII) activity has been used in the study of 35 patients with severe chronic hepatopathy, in comparison with 25 normal subjects. The FXIII proteic fractions a and b were determined by quantitative immuno-electrophoresis after Laurell. The plasmatic FXIII activity, as well as the proteic fractions a and b, were significantly reduced in hepatopatic patients, in comparison to controls, and proportional to the prolongation of prothrombin times. Ratios between functional and immunological levels of FXIII in hepatopatics were similar to those observed in controls. These results confirm the involvement of fibrin stabilization deficiency in the coagulation defect of severe chronic hepatopathies. The correlations between functional and antigenic values are in agreement with the hepatic origin of FXIII. The method of Fickenscher has been proved to be rapid and simple, and it may be useful in the routine study of hepatopathies, for a better knowledge of the role of FXIII deficiency in the complex coagulopathy of liver diseases, as well as of other acquired FXIII deficiencies.

Non-haemorrhagic adverse reactions of oral anticoagulant therapy.

Oral anticoagulants are extensively used in everyday medical practice, especially for the prophylaxis of deep vein thrombosis and pulmonary thromboembolism. Bleeding is the major risk of such therapy. Although infrequent, however, non-haemorrhagic complications may also play a considerable role. The purpose of this paper is briefly to review the most important non-haemorrhagic adverse reactions and their clinical signs. Moreover, the pathogenetic hypotheses, the relationships with protein C and S levels, and the possibility of prevention and treatment are also discussed.

Coexistence of antithrombin deficiency, factor V Leiden and hyperhomocysteinemia in a thrombotic family.

We report a thrombotic family with combined type I antithrombin deficiency and factor V Leiden (factor V-R506Q) in which the proposita, affected by recurrent venous and arterial thrombosis, was also characterized by mild hyperhomocysteinemia (28 micromol/l; normal <18.5 micromol/l). Her two thrombotic sisters, with normal antithrombin levels and factor V molecules, showed hyperhomocysteinemia (51 and 30 micromol/l, respectively). Four other members of the family had the combined antithrombin/factor V Leiden defect and two of them had thrombosis. The common A223V mutation in the methylenetetrahydrofolate reductase gene, responsible for the thermolabile variant of the enzyme, was found to be heterozygous in the proposita; the two sisters were homozygous and heterozygous, respectively. The heterozygous sister also had a high titre of antiphospholipid antibodies (85 units of immunoglobulin G antiphospholipid antibody/ml). Furthermore, low plasma folate levels were found in the three hyperhomocysteinemic subjects of the family. This family with several prothrombotic defects is a clear example of the polyfactorial nature of thrombophilia.

Resistance to activated protein C and low levels of protein S activity in nine thrombophilic families: a correct diagnosis.

In order to define the thrombophilic conditions related to activated protein C resistance (APC-R) and protein S (PS) deficiency and to detect the possible combination of these defects, we studied nine unrelated patients selected because of low anticoagulant response to APC and reduced PS activity with at least one first degree relative having the same coagulation feature. The mean APC ratio was 0.64 (normal values > 0.80) and range 0.35-0.80. Three of the patients were heterozygous and two were homozygous for the Leiden mutation (FVR506Q); in the remaining four there was no mutation. The mean PS activity was 41.6% and range 32-54 (normal 65-150%). Five of the patients had low PS activity despite normal total and free antigenic levels, and normal activity when measured at higher plasma dilution; these were carrying at least one gene for the Leiden mutation. In the remaining four patients the crossed-immunoelectrophoresis and the Western-blotting analysis showed a type I PS deficiency confirmed by the familial restriction fragment length polymorphism analysis. Thus, four families were diagnosed with the type I PS defect and five with congenital APC-R. No combined PS/FV Leiden or type II PS defect was found. The only defect found was in the anticoagulant PC pathway. We therefore designed a procedure to diagnose thrombophilic conditions related to this pathway. This study indicates that a specific methodological approach must be used to accurately characterize APC-R and PS deficiency and that care is necessary to avoid the possibility of misdiagnosis.

The reduced sensitivity of the ProC Global test in protein S deficient subjects reflects a reduction in the associated thrombotic risk.

To investigate simultaneously a defect affecting the protein C/protein S (PC/PS) anticoagulant pathway is possible thanks to a methodological approach (ProC(R) Global; Dade Behring) based on the activation of endogenous plasma PC by a snake venom extract. Factor V (FV) Leiden, the most frequent cause of hereditary thrombosis, is well detected by the test with sensitivity of 100% irrespective of the presence/absence of thrombosis in the subjects investigated. The test is also suited to detect PC or PS defect, but in this case the in vitro impairment of the PC/PS pathway is less pronounced particularly for PS defects (sensitivity for PC and PS defect, 85-100 and 30-90%, respectively). In this study, we hypothesized that the lower sensitivity described for PS defect, compared with those of PC and FV Leiden defects, could also be related to the clinical condition of the subject investigated (symptomatic/asymptomatic) rather than solely to the PS plasma activity/level. Therefore, we analyzed 126 subjects with single congenital defects in the PC/PS pathway: 46 subjects with PS deficiency (26 thrombotic cases and 20 asymptomatic relatives), 40 subjects with PC deficiency (25 thrombotic cases and 15 asymptomatic relatives), and 40 heterozygous FV Leiden subjects (25 thrombotic cases and 15 asymptomatic relatives). By a cut-off of normalized Agkistrodon contortix snake venom ratio of 0.84, the sensitivity in the whole group of cases (sensitivity a) was 76.1, 95.0 and 100%, respectively, for PS, PC and FV Leiden defects. The test failed to detect 11 (23.9%) among the 46 PS-deficient subjects, and all these cases except two belonged to the asymptomatic subgroup (9/20; 45%). Excluding the 20 asymptomatic relatives, the new sensitivity (sensitivity b) for the PS defect was 92.3%. The comparison of the sensitivity in the symptomatic PS cases and in the asymptomatic ones was significantly different (P = 0.010). Among the 40 PC-deficient subjects, only two (5.0%) were not detected by the test and they belonged indifferently to the two subgroups. Finally, none of the 40 FV Leiden heterozygotes were misdiagnosed by the test. These results suggest that in symptomatic PS-deficient cases the test could reflect a post-thrombotic effect and/or reveal potential unidentified prothrombotic influences assessing a prothrombotic risk condition.

Thrombotic risk in thalassemic patients.

Hemostatic parameters of 495 beta-thalassemic patients (421 with thalassemia major and 74 with thalassemia intermedia) were analyzed, to assess their association with the described thrombophilic condition and to verify the role of additional risk factors (e.g. persistent postsplenectomy thrombocytosis, insulin dependent diabetes mellitus, estrogen-progestin treatment and atrial fibrillation). The prevalence of thromboembolic accidents was 5.2% and in four patients (15.3%) inherited or acquired predisposing defects were recognized. The incidence of thromboembolic events and the associated relative risk due to hemocoagulative abnormalities in these patients are discussed.

Anticardiolipin antibody-related thrombocytopenia: persistent remission after splenectomy.

The lupus anticoagulant (LAC) and anticardiolipin antibody (ACA) syndromes require particular therapeutic approaches: thrombotic accidents are an indication for oral anticoagulant therapy (OAT), whereas severe thrombocytopenia may require the special treatments used for immunologic thrombocytopenic purpura (ITP). We describe the case of a 21-year-old male who presented with axillary vein thrombosis associated with LAC and ACA at high titers in December 1990. OAT was begun and, due to repeated episodes of thrombocytopenia, high-dose steroid therapy was later added with success. The daily steroid dose was reduced because of patent hypercortisolism, but the platelet count fell to 4 x 10(9)/L. A bone marrow biopsy was characteristic for ITP. Splenectomy was performed in June 1993, and the platelet count rapidly normalized. Platelet antibodies were always detectable before and after splenectomy. The patient is currently asymptomatic, with platelet counts above 300 x 10(9)/L at one and a half years after splenectomy. This case indicates that ACA-associated thrombocytopenia, like ITP and HIV-related thrombocytopenias, can be successfully treated with steroids and splenectomy, even though different pathogenetic mechanisms are responsible for the antibody-induced platelet consumption.

Long-lasting remission and successful treatment of acquired factor VIII inhibitors using cyclophosphamide in a patient with systemic lupus erythematosus.

Acquired deficiency of clotting factor VIII (FVIII) is a rare bleeding diathesis seldom encountered in systemic lupus erythematosus (SLE). Reduction of FVIII activity by autoantibodies can cause potentially life-threatening situations. Herein, an SLE patient with a positive lupus anticoagulant (LAC) test who abruptly developed metrorrhagia 4 yr after diagnosis is reported. Coagulation tests revealed FVIII activity reduced to 3% and a prolonged aPTT. FVIII inhibitor(s) were found to be as high as 3.0 Bethesda Units. Plasmapheresis, immunoglobulins, prednisolone and FVIII plasma concentrates induced the cessation of metrorrhagia, but the clotting tests were barely improved. One month later, extensive ecchymosis appeared and worsened, despite re-administration of the previous therapy. Pulse cyclophosphamide followed by oral administration was then started with normalization of coagulation parameters and long-lasting disease remission.

Coexistence of factor V G1691A and factor II G20210A gene mutations in a thrombotic family is associated with recurrence and early onset of venous thrombosis.

Two G-to-A mutations at positions 1691 of the factor V (FV) gene and 20210 of the prothrombin (FII) gene have been associated with an increased risk of venous thromboembolism. We report a thrombosis-prone family in which one subject--the propositus who exhibited combined heterozygous FV G1691A and FII G20210A mutations--showed spontaneous and early clinical onset (at 23 years), recurrences of deep-vein thrombosis and pulmonary embolism. His asymptomatic father carried the FII G20210A substitution and his mother, characterized by an isolated thrombotic episode on occasion of surgery (at 48 years), carried the FV G1691A substitution. In the maternal lineage, one of the propositus' uncles had thrombosis on occasion of a bone fracture (at 65 years) despite the absence of known prothrombotic defects. A sister of the propositus carried the FII G20210A and the brother the FV G1691A mutation. They have been asymptomatic until now. The propositus' two children, 20 and 16 years old, both carry the FV G1691A substitution and have been asymptomatic until now. The plasma levels of FII were higher in carriers of the FII G20210A allele if compared with noncarriers, and the activated protein C resistance phenotype, associated with the FV Leiden mutation, showed a complete correlation with the FV G1691A mutation. Despite the very limited number of thrombotic cases involved in this survey, which does not allow statistically sound conclusions, the data obtained from this family suggest that the synergy of inherited factors and transient risk conditions could play a key role in the occurrence of thrombotic accidents.

A novel mutation (Leu817Pro) causing type 2A von Willebrand disease.

We studied a patient affected by von Willebrand disease type 2A who experienced several mild bleeding episodes and was characterized by markedly reduced haemostatic parameters. In the exon 28 of von Willebrand factor (vWF) gene a T to C transition at nucleotide 8680, resulting in the missense mutation Leu817Pro, was found in the heterozygous form in the patient and in two affected relatives. As suggested by the presence in platelets of a complete spectrum of vWF multimers as well as by the increased vWF antigen levels and improved haemostasis after DDAVP treatment, the mutation is compatible with normal multimerization, and could be responsible for a reduced stability or an impaired physiological secretion of vWF.

A common mutation in the gene for coagulation factor XIII-A (VAL34Leu): a risk factor for primary intracerebral hemorrhage is protective against atherothrombotic diseases.

The role of a common polymorphism in the factor XIII A-subunit gene (FXIII Val34Leu) has been recently investigated as a protective genetic factor against arterial and venous thrombosis. In addition, the less frequent Leu34 allele has been described as a risk factor for intracerebral hemorrhage. We evaluated the prevalence of this polymorphism by PCR in three case-control studies of patients diagnosed as having primary intracerebral hemorrhage (PCH, n = 130), coronary heart diseases (CHD, n = 240; myocardial infarction/no myocardial infarction, 120/120), and cerebrovascular diseases (CVD, n = 240; cerebral infarction/transient ischaemic attack, 120/120). The matched control groups consisted of patients admitted to the hospital without history of vascular disease. In addition, 200 healthy subjects were investigated. The frequency of the mutated allele (Leu34) was higher in patients with PCH than in controls (33.8% vs. 23.1%, P = 0.009) and lower in CHD and CVD patients compared to controls (18.1% vs. 25.2%, P = 0.010 and 17.3% vs. 24.2%, P = 0.011, respectively). Moreover, among the patients with CHD, the Leu34 allele was underrepresented in cases with myocardial infarction than without (12.9% vs. 23.3%, P = 0.004) and than in controls (12.9% vs. 25.2%, P < 0.001). Similar findings were obtained in patients with CVD comparing the cases with cerebral infarction versus cases with transient ischaemic attack (12.5% vs. 22.1%, P = 0.008) and versus controls (12.5% vs. 24.2%, P < 0.001). Finally, considering altogether the groups of ischaemic patients (CHD and CVD, n = 480), it was noted a trend towards a higher mean age of the clinical onset in homozygotes for the Leu allele than in the wild types (P = 0.078). This study indicates that in our population possession of the FXIII Val34Leu mutation predisposes to the occurrence of primary intracerebral hemorrhage and protects against cerebral and myocardial infarction. A wider modulatory role in the progression and onset of atherothrombotic diseases could be ascribed to FXIII Val34Leu.

Pattern of iron excretion in relation to haemoglobin level and iron load in 8 haematological patients following the administration of subcutaneous deferrioxamine.

Iron excretion following subcutaneous administration of deferrioxamine (DFO) was measured between two transfusions of packed red cells in 6 patients with beta-thalassaemia major on the high level Hb transfusion regime; and in a single 3-day period in 2 other patients, 1 with transfused beta-thalassaemia major and the other with haemolytic anaemia due to PK deficiency. The pattern of iron excretion did not change significantly during the period between the two transfusions and was found to be related to serum ferritin levels. The proportion of iron excreted in the stools was inversely related to the serum ferritin level. These observations on iron excretion are of practical importance in relation to DFO administration, especially when evaluated in thalassaemics with normal haemoglobin levels and low iron stores.

Low folate levels and thermolabile methylenetetrahydrofolate reductase as primary determinant of mild hyperhomocystinemia in normal and thromboembolic subjects.

Several studies have indicated that mild to moderate hyperhomocystinemia is a common cause of arterial occlusive disease. Whether hyperhomocystinemia per se is an independent risk factor for vein thromboembolism (VTE) is still somewhat controversial. Both genetic and nutritional factors influence plasma homocysteine levels. Therefore, we evaluated plasma total homocysteine (tHcy), folate, and vitamin B12 levels and established, by polymerase chain reaction, the presence of the C677T mutation (A223V) in the methylenetetrahydrofolate reductase (MTHFR) gene in 220 cases with VTE without well-established prothrombotic defects. As a control group, 220 healthy subjects from the same geographic area as the cases were investigated. Hyperhomocystinemia was defined as a plasma tHcy level above the 95th percentile in the controls (18.05 micromol/L). Hyperhomocystinemia was found in 16% of cases (odds ratio=3.59; P<0.001); deficiencies of folate (<2.47 ng/mL) or vitamin B12 (<165 pg/mL), defined as values below the 5th percentile in controls, were found in 17.7% (P<0.001) and 12.3% (P=0.015) of cases, respectively. The homozygous condition for the MTHFR mutation (VV) was present in 28.2% of cases and 17.7% of controls (odds ratio=1.82; P=0.013). Comparing only the idiopathic forms of VTE (n=80/220; 36.3%) with normal controls, individuals with hyperhomocystinemia, or individuals homozygous for MTHFR mutation increased the odds ratios to 4.03 (P=0.005) and 2.11 (P=0.018), respectively. No statistically significant difference was observed in the MTHFR genotype distribution of cases and controls with hyperhomocystinemia (P=0.386); however, the normal MTHFR genotype (AA) appeared in control subjects only when tHcy levels were below the 80th percentile (10.57 micromol/L) of the distribution, whereas in case patients, it was present at the highest tHcy levels. A strong association between mutated homozygosity (VV), low folate levels, and hyperhomocystinemia was found in both groups. We conclude that in patients with VTE who do not have coexisting prothrombotic defects, hyperhomocystinemia increases the risk of developing idiopathic and venous thrombosis; the homozygous condition for the MTHFR mutation confers a moderate risk but, together with low folate levels, it is the main determinant of mild hyperhomocystinemia in normal and thromboembolic populations.