"In vitro" correction of the severe factor V deficiency-related coagulopathy by a novel plasma-derived factor V concentrate.

INTRODUCTION: Severe congenital factor V (FV) deficiency is a rare bleeding disorder characterized by very low/undetectable levels of FV. Fresh frozen plasma is the standard treatment for bleeding manifestations. Recently, a novel plasma-derived FV concentrate has been developed. AIM: To evaluate the "in vitro" ability of the novel FV concentrate to normalize clotting times and generate normal amount of thrombin in plasma collected from patients with severe FV deficiency. METHODS: Prothrombin time (PT), activated partial thromboplastin time (aPTT), FV activity and antigen levels and thrombin generation were measured pre- and postspiking of plasma samples of 10 patients with increasing doses of FV concentrate (from 0 to 100 IU/dL). RESULTS: Prothrombin time and activated partial thromboplastin time ratios as well as all thrombin generation parameters were fully corrected by the addition of FV concentrate at a final concentration of 25 IU/dL. However, the addition of FV at a concentration of 1-3 IU/dL was already sufficient to correct peak height and endogenous thrombin potential (but not lag time and time to peak) after activation with 5 pmol/L tissue factor. FV activity and antigen levels showed a linear response to supplementation with the novel FV concentrate. CONCLUSION: The novel plasma-derived FV concentrate was effective to correct "in vitro" severe FV deficiency in patients. The optimal FV concentration to fully normalize both global clotting times and thrombin generation parameters using the novel plasma-derived FV concentrate was 25 IU/dL.

Multiplex ligation-dependent probe amplification as first mutation screening for large deletions and duplications in haemophilia.

INTRODUCTION: Molecular characterization has shown a wide mutational spectrum underlying haemophilia A (HA) and haemophilia B (HB). Different molecular assays have allowed laboratories to perform genetic testing for F8 and F9 mutations. AIM: Recently, multiplex ligation-dependent probe amplification (MLPA), a simple technique for relative quantitation of targeted genomic regions, has been introduced in HA and HB for detection of large deletions and duplications. We want to verify if MLPA might be used at the beginning of the molecular investigation. METHODS: We used it to test 22 patients with suspected large deletions, nine patients negative for mutation detection by other methods and finally, 45 new patients as their first screening test. RESULTS: Carrier status was also established in 28 related females and gross rearrangements were also searched for by MLPA in 19 females with reduced FVIII or FIX levels. All suspected deletions were confirmed apart from two patients. In patients with a negative screening test, MLPA revealed one large duplication, while in two patients where MLPA was used as the first screening step, an exon duplication was detected. In females with reduced FVIII or FIX, no large deletions or duplications were found. CONCLUSIONS: Owing to its simplicity, MLPA seems useful at the beginning of the molecular investigation, saving all the following steps, where positive. Single exon deletion diagnosis requires caution due to the risk of misdiagnosis, but benefits of MLPA appear to overcome the pitfalls.

Correlation between FIX genotype and pharmacokinetics of Nonacog alpha according to a multicentre Italian study.

INTRODUCTION: Pharmacokinetic (PK) studies on recombinant FIX concentrate, Nonacog alpha, were conducted with different sampling time designs which gave rise to not complete and homogenous outcomes. In addition, patient's FIX genotype/PK relationship has never been investigated. AIM: Investigate how different sampling times may affect PK parameters and try to find a FIX genotype/PK relationship. PATIENTS AND METHODS: A cohort pharmacokinetic, Nonacog Alpha single-dose, open-label, non-comparative study was conducted in eight Comprehensive Care Haemophilia Centres in Italy. Seventeen previously treated moderate or severe haemophilia B patients were enrolled. Factors IX:C one-stage clotting assay, FIX genotype and PK analysis were centralized. RESULTS: The evaluation of PK outcomes showed a quite long half-life, smaller clearance and volume of distribution of Nonacog Alpha in comparison with the results from previously reported studies, where blood sampling was stopped too early. The relationship between PK outcomes and FIX genotype showed that small deletions displayed the higher clearance and shorter half-life, the nonsense mutations (the lower and the longer respectively), and missense mutations were in between. CONCLUSIONS: It is evident that area under the curve (AUC) and other PK parameters depend from the sampling time design. In order to have a complete evaluation of clotting factors in vivo decay, blood samples must be collected until the baseline factor concentration has been achieved again. Due to the relationship between FIX genotype and clearance, tailored prophylaxis of HB patients could be partially predicted by genotyping.

Inhibitors in haemophilia B: the Italian experience.

The prevalence of inhibitors in haemophilia B is significantly lower than that of patients with haemophilia A. However, the peculiar occurrence of allergic reactions associated with the onset of inhibitor in haemophilia B (HB) may render immune tolerance a risky procedure. We have carried out a detailed survey among all the Italian Hemophilia Centers to analyse all the patients with HB and inhibitors. A total of eight patients were reported among 282 living patients (2.8%) with severe factor IX (FIX) deficiency (FIX < 1 U dL(-1)). In addition, two deceased patients were also identified. Six patients carried nonsense mutations while in four partial or complete gene deletions were detected. Three patients (one deceased) had history of allergic/anaphylactic reaction upon substitutive treatment, which in one case was recurrent and resolved after switching to plasma derived FIX. Immune tolerance was adopted in five patients and in four complete response was achieved while in the remaining it was partial. No nephrotic syndrome was observed. Our data confirm that inhibitors in HB occur in patients with null mutations or complete/partial gene deletion. Immune tolerance can be achieved also in HB patients, without allergic reactions or nephrotic syndrome upon replacement therapy.

Prenatal diagnosis of haemophilia B: the Italian experience.

This article describes prenatal diagnosis (PND) of haemophilia B (HB) within the framework of Italian haemophilia centres and genetics laboratories. The study details the experience from six haemophilia genetic centres (three in the North, one in the Centre and two in the South of Italy) and summarizes the different techniques used to perform PND of HB during the last 15 years. To date, the Italian HB database includes 373 characterized unrelated patients and their genetic information has permitted the identification of 274 carriers of childbearing age. This database represents the main instrument for timely and precise PND. Sixty-six prenatal diagnoses were performed on 52 HB carriers whose average age at the time was 34 (ranging from 24 to 44 years). In 44 cases, genetic counselling for carrier status determination was performed before pregnancy, while eight were not studied prior to pregnancy. Foetal samples were obtained by chorionic villus sampling in 52 cases, by amniocentesis in 12 while two were diagnosed by analysis of free foetal DNA obtained from maternal peripheral blood. In 35 (53%) pregnancies the foetus was female. For 31 men (47%), haemophilia status was determined by analysis of previously determined informative markers or familial mutations (12 affected and 19 unaffected). There may be more than one laboratory involved in the PND diagnostic pathway (providing DNA extraction, karyotype analysis, gender determination, maternal contamination detection, molecular diagnosis and sequencing). Good communication between all the parties, coordinated by the haemophilia centre, is essential for a successful and rapid process.

Rehabilitation of patients with thin ridges by conical expanders and immediate cone morse dental implant: a case report.

BACKGROUND: Horizontal atrophic ridges need a regenerative procedure for implant positioning and fixed rehabilitation. Cone Morse taper implants are characterized by the intimate fitting of the prosthetic interface with the absence of microgaps and micromovements of the interfaces. The aim of this case report was to evaluate the clinical outcome of Cone Morse implant design in split crest augmentation treatment. CASE REPORT: A female patient with partial edentulism of atrophic posterior maxilla was treated for split crest procedure and implant-supported rehabilitation. A full-thickness flap was elevated, and horizontal and vertical osteotomic lines were produced with piezoelectric device. A total of 4 Cone Morse Taper implants (Universal III, Implacil de Bortoli, Brasil) were positioned and the site was grafted with bone substitute and covered by a heterologous membrane. CONCLUSIONS: A complete healing of the surgical site was evident at the follow-up with no evidence of bone resorption. No radiolucency or inflammatory aspects of the treated site were evident in the radiographic control. Simultaneous Cone Morse implants positioning with split crest technique seems to be a promising treatment for posterior maxillary rehabilitation of atrophic edentulous ridges.

Topical hemostatic agents in oral surgery: a narrative review.

Sufficient hemostasis during oral surgical procedures is crucial for successful outcomes and to reduce healthcare resource utilization. The purpose of this narrative review is to give a rational insight into the management of bleeding in oral and dental practice through modern drugs. A narrative literature review has been performed on the present topic identifying all articles on Pubmed/Medline and Google Scholars. Acceptable hemostasis during oral surgery is also required to improve visibility and provide a dry operational area. Many oral surgeons, in their daily practice, encounter problems in controlling postoperative bleeding and use a topical hemostatic agent to promote platelet activation or aggregation to form a stable clot.

Polymorphisms in genes involved in autoimmune disease and the risk of FVIII inhibitor development in Italian patients with haemophilia A.

One of the most severe and important complication in the treatment of patients with haemophilia A is the formation of neutralizing antibodies (FVIII inhibitors) that inhibit the clotting activity of substituted FVIII. Both genetic and environmental factors influence the susceptibility of patients to develop inhibitors. The objective of this study was to evaluate whether polymorphisms in different genes involved in the regulation of the immune system may confer susceptibility to inhibitor development in patients with HA. We analysed the distribution of polymorphisms in the CTLA4, PTPN22, IL10, TNFalpha, FOXP3 and IRF5 genes that have been reported to be associated with a number of autoimmune disease. In addition, we evaluated the distribution of IL10 haplotypes in haemophilic patients and healthy controls to assess whether specific polymorphisms in IL10 gene were associated to the risk of inhibitor development. We focused on a cohort of Italian unrelated haemophilic patients with and without a history of inhibitors. Genotyping was carried out with standard methods including RFLP, real time PCR and direct DNA sequencing. Our data show that, considering single nucleotide variations, genotype frequencies in patients with inhibitors were not significantly different from those observed in patients without inhibitors, suggesting a lack of association between these polymorphisms and the development of inhibitors. Moreover, no relationship was found between specific combinations of IL10 alleles and the antibody production. Previous contradictory association studies may depend on the different genetic background of the population examined. Further studies may contribute to a clearer understanding of this process.

Mortality and causes of death in Italian persons with haemophilia, 1990-2007.

Although a number of studies have analysed so far the causes of death and the life expectancy in haemophilic populations, no investigations have been conducted among Italian haemophilia centres. Thus, the aim of this study was to investigate mortality, causes of deaths, life expectancy and co-morbidities in Italian persons with haemophilia (PWH). Data pertaining to a total of 443 PWH who died between 1980 and 2007 were retrospectively collected in the 30 centres who are members of the Italian Association of Haemophilia Centres that chose to participate. The mortality rate ratio standardized to the male Italian population (SMR) was reduced during the periods 1990-1999 and 2000-2007 such that during the latter, death rate overlapped that of the general population (SMR 1990-1999: 1.98 95% CI 1.54-2.51; SMR 2000-2007: 1.08 95% CI 0.83-1.40). Similarly, life expectancy in the whole haemophilic population increased in the same period (71.2 years in 2000-2007 vs. 64.0 in 1990-1999), approaching that of the general male population. While human immunodeficiency virus infection was the main cause of death (45%), 13% of deaths were caused by hepatitis C-associated complications. The results of this retrospective study show that in Italian PWH improvements in the quality of treatment and global medical care provided by specialized haemophilia centres resulted in a significantly increased life expectancy.

Phase 1, single-dose escalating study of marzeptacog alfa (activated), a recombinant factor VIIa variant, in patients with severe hemophilia.

Essentials Marzeptacog alfa (activated) [MarzAA] is a novel variant of activated human factor VII. A phase 1 dose escalation trial of MarzAA was conducted in subjects with severe hemophilia. MarzAA was safe and tolerated at intravenous doses up to 30 µg kg-1 Data observed support further trials for hemophilia patients with inhibitors to factors VIII/IX. SUMMARY: Background Marzeptacog alfa (activated) (MarzAA), a new recombinant activated human factor VII (rFVIIa) variant with four amino acid substitutions, was developed to provide increased procoagulant activity and a longer duration of action in people with hemophilia. Objectives To investigate the safety, tolerability, immunogenicity, pharmacokinetics (PK) and pharmacodynamics (PD) of single ascending intravenous bolus doses of MarzAA in non-bleeding patients with congenital hemophilia A or B with or without inhibitors. Methods This international, phase 1, open-label study (NCT01439971) enrolled males aged 18-64 years with severe hemophilia A or B, with or without FVIII or FIX inhibitors. Subjects were assigned to single-dose MarzAA cohorts (0.5, 4.5, 9, 18 or 30 µg kg-1 ). Blood sampling was performed predose and postdose, and subjects were monitored for 60 days postdose. Safety endpoints included adverse events, vital sign changes, electrocardiograms, laboratory abnormalities, and immunogenicity; secondary endpoints included evaluation of PK and PD. Results Overall, in 25 patients, MarzAA was well tolerated at all dose levels tested, and was not associated with dose-limiting toxicity. No treatment-emergent severe or serious adverse events occurred. MarzAA showed linear dose-response PK across the 4.5-30 µg kg-1 dose range, with a terminal half-life of ⁓ 3.5 h. Dose-dependent shortening of the activated partial thromboplastin time and prothrombin time, and evidence of an increase in peak thrombin as determined with a thrombin generation assay, were observed at all doses. Conclusions MarzAA was tolerated at doses up to 30 µg kg-1 . The safety profile and pharmacological effects observed support further clinical trials for the treatment of hemophilic patients with inhibitors.

Clinical factors associated with progression to AIDS in the Italian cohort of HIV-positive hemophiliacs. G.I.C.C. Gruppo Italiano Coagulopatie Congenite.

This study updates estimates of the cumulative incidence of AIDS among Italian patients with congenital coagulation disorders (mostly hemophiliacs), and elucidates the role of age at seroconversion, type and amount of replacement therapy, and HBV co-infection in progression. Information was collected both retrospectively and prospectively on 767 HIV-1 positive patients enrolled in the on-going national registry of patients with congenital coagulation disorders. The seroconversion date was estimated as the median point of each patient's seroconversion interval, under a Weibull distribution applied to the overall interval. The independence of factors associated to faster progression was assessed by multivariate analysis. The cumulative incidence of AIDS was estimated using the Kaplan-Meier survival analysis at 17.0% (95% CI = 14.1-19.9%) over an 8-year period for Italian hemophiliacs. Patients with age greater than or equal to 35 years exhibited the highest cumulative incidence of AIDS over the same time period, 32.5% (95% CI = 22.2-42.8%). Factor IX recipients (i.e. severe B hemophiliacs) had higher cumulative incidence of AIDS (23.3% vs 14.2%, p = 0.01) than factor VIII recipients (i.e. severe A hemophiliacs), as did severe A hemophiliacs on less-than-20,000 IU/yearly of plasma-derived clotting factor concentrates, as opposed to A hemophiliacs using an average of more than 20,000 IU (18.8% vs 10.9%, p = 0.02). No statistically significant difference in progression was observed between HBsAg-positive vs HBsAg-negative hemophiliacs (10.5% vs 16.4%, p = 0.10). Virological, immunological or both reasons can account for such findings, and should be investigated from the laboratory standpoint.

Markers of hypercoagulability in patients with hemophilia B given repeated, large doses of factor IX concentrates during and after surgery.

Purer factor IX (FIX) concentrates have been produced for the treatment of hemophilia B in the attempt to reduce the risk of thrombotic complications associated with the use of prothrombin complex concentrates. To evaluate ex vivo whether or not FIX concentrates activate the coagulation system in conditions associated with a high risk for thrombosis, we measured markers of hypercoagulability in 10 patients with hemophilia B who underwent surgery, mainly orthopedic procedures, covered by multiple concentrate infusions (40-80 U/kg/day). Postinfusion plasma levels of prothrombin fragment 1 + 2 and factor X activation peptide did not differ significantly from the presurgical levels, neither before nor after each concentrate dose. Therefore, it appears that prolonged treatment of patients with hemophilia B undergoing high risk surgical procedures with high doses of FIX concentrate does not cause systemic activation of coagulation. This suggests that purified FIX concentrates are preferable to prothrombin complex concentrates for conditions associated with an increased risk of thrombosis.

A rapid and cost effective method for analysis of dinucleotide repeat polymorphisms in the factor VIII gene.

A simple, rapid and cheap method for the analysis of variable dinucleotide tandem polymorphisms in introns 13 and 22 of the factor VIII gene has been established. The protocol uses blood spots stored on filter paper (Guthrie spots) and other DNA containing materials as sources of DNA. Following DNA amplification using a thermostable DNA polymerase, products are size fractionated on native polyacrylamide gels and visualized by silver staining. This simplified protocol obviates the use of DNA extraction, reducing time and costs and in addition, reduces the requirement for gel documentation equipment (i.e., photography), as silver staining can be visualized readily without additional equipment and the gels themselves can be stored. These alterations to the analysis enhance the universal applicability of these polymorphisms, as was demonstrated by a comparative study in Caucasian and Thai females.

Serological markers of autoimmunity in patients with hemophilia A: the role of hepatitis C virus infection, alpha-interferon and factor VIII treatment in skewing the immune system toward autoreactivity.

The aim of this study was to investigate whether the immune system of patients with hemophilia A is skewed toward an aspecific activation and to identify the causative factors. It is well known that an immune derangement does exist in patients with hemophilia A. At least three factors potentially play a role: hepatitis C virus (HCV) infection, alpha-interferon therapy and the administration of factor VIII (FVIII). Sixty human immunodeficiency virus (HIV)-negative patients with severe or moderate hemophilia A were studied retrospectively. The serological markers of autoimmunity were evaluated and the results correlated with anti-HCV antibodies, FVIII treatment and alpha-interferon therapy. The role of these factors in the development of the anti-FVIII antibody was estimated concomitantly. The prevalence of autoantibodies and anti-FVIII antibodies was higher in HCV-positive than in HCV-negative patients before any treatment, although the difference was not statistically significant. The administration of FVIII further influenced the development of autoantibodies both in HCV-negative and HCV-positive patients, with no difference being observed between the two groups. As expected, fewer HCV-negative than HCV-positive patients developed anti-FVIII antibodies after administration of FVIII (31.8% versus 38%, respectively). Therapy with alpha-interferon did not seem to enhance significantly the risk of developing autoantibodies nor anti-FVIII antibodies. We observed a high prevalence of humoral signs of autoimmunity among patients with hemophilia A. Treatment with FVIII concentrate is probably the most important triggering factor. Monitoring these patients for autoimmune manifestations is recommended.

Homozygous F5 deep-intronic splicing mutation resulting in severe factor V deficiency and undetectable thrombin generation in platelet-rich plasma.

BACKGROUND: Coagulation factor (F) V deficiency is associated with a bleeding tendency of variable severity, but phenotype determinants are largely unknown. Recently, we have shown that three patients with undetectable plasma FV and mild bleeding symptoms had sufficient residual platelet FV to support thrombin generation in platelet-rich plasma (PRP). Therefore, we hypothesized that FV-deficient patients with severe bleeding manifestations may lack platelet FV. OBJECTIVES: To characterize a FV-deficient patient with a severe bleeding diathesis. PATIENTS/METHODS: We performed FV mutation screening and functional studies in a 31-year-old male (FV:C < 1%) with umbilical bleeding at birth, recurrent hemarthrosis and muscle hematomas, and a recent intracranial hemorrhage. RESULTS: The proband was homozygous for a deep-intronic mutation (F5 IVS8 +268A→G) causing the inclusion of a pseudo-exon with an in-frame stop codon in the mature F5 mRNA. Although platelet FV antigen was detectable by immunoprecipitation followed by Western blotting, no FV activity could be demonstrated in the proband's plasma or platelets with a prothrombinase-based assay. Moreover, no thrombin generation was observed in PRP triggered with 1-50 pm tissue factor (even in the presence of platelet agonists), whereas an acquired FV inhibitor was excluded. Clot formation in the proband's whole blood, as assessed by thromboelastometry, was markedly delayed but not abolished. CONCLUSIONS: This is the first report of a pathogenic deep-intronic mutation in the F5 gene. Our findings indicate that the minimal FV requirement for viability is extremely low and suggest that thrombin generation in PRP may predict bleeding tendency in patients with undetectable plasma FV.