Angelo Claudio Molinari

Factor VIII Products and Inhibitor Development in Severe Hemophilia A

BACKGROUND For previously untreated children with severe hemophilia A, it is unclear whether the type of factor VIII product administered and switching among products are associated with the development of clinically relevant inhibitory antibodies (inhibitor development). METHODS We evaluated 574 consecutive patients with severe hemophilia A (factor VIII activity, <0.01 IU per milliliter) who were born between 2000 and 2010 and collected data on all clotting-factor administration for up to 75 exposure days. The primary outcome was inhibitor development, which was defined as at least two positive inhibitor tests with decreased in vivo recovery of factor VIII levels. RESULTS Inhibitory antibodies developed in 177 of the 574 children (cumulative incidence, 32.4%); 116 patients had a high-titer inhibitory antibody, defined as a peak titer of at least 5 Bethesda units per milliliter (cumulative incidence, 22.4%). Plasma-derived products conferred a risk of inhibitor development that was similar to the risk with recombinant products (adjusted hazard ratio as compared with recombinant products, 0.96; 95% confidence interval [CI], 0.62 to 1.49). As compared with third-generation full-length recombinant products (derived from the full-length complementary DNA sequence of human factor VIII), second-generation full-length products were associated with an increased risk of inhibitor development (adjusted hazard ratio, 1.60; 95% CI, 1.08 to 2.37). The content of von Willebrand factor in the products and switching among products were not associated with the risk of inhibitor development. CONCLUSIONS Recombinant and plasma-derived factor VIII products conferred similar risks of inhibitor development, and the content of von Willebrand factor in the products and switching among products were not associated with the risk of inhibitor development. Second-generation full-length recombinant products were associated with an increased risk, as compared with third-generation products. (Funded by Bayer Healthcare and Baxter BioScience.).

Development and definition of a simplified scanning procedure and scoring method for Haemophilia Early Arthropathy Detection with Ultrasound (HEAD-US)

The aim of this study was to develop a simplified ultrasound scanning procedure and scoring method, named Haemophilia Early Arthropathy Detection with UltraSound [HEAD-US], to evaluate joints of patients with haemophilic arthropathy. After an initial consensus-based process involving a multidisciplinary panel of experts, three comprehensive and evidence-based US scanning procedures to image the elbow, knee and ankle were established with the aim to increase sensitivity in detection of early signs of joint involvement while keeping the technique easy and quick to perform. Each procedure included systematic evaluation of synovial recesses and selection of a single osteochondral surface for damage analysis. Based on expert consensus, a simplified scoring system based on an additive scale was created to define the joint status and, in perspective, to offer a tool to evaluate disease progression and monitor the result of treatment in follow-up studies.

Prospective study of hemostatic alterations in children with acute lymphoblastic leukemia

In a group of newly diagnosed acute lymphocytic leukemia (ALL) children we evaluated a number of hemostatic and inflammatory markers at diagnosis and at different time points during chemotherapy for the remission induction to identify alterations in the plasma levels of prothrombotic markers before and during the course of chemotherapy. The following plasma markers were evaluated: thrombin‐antithrombin complex (TAT), D‐Dimer, plasminogen activator inhibitor 1 (PAI‐1), antithrombin, fibrinogen, von Willebrand factor (VWF) antigen and high molecular weight VWF (HMW‐VWF) multimers, P‐selectin, tumor necrosis factor alpha (TNF‐α), and interleukin 6 (IL‐6). Plasma samples were collected at the following time points: at T0 (baseline) and T1 (+24 days of therapy), T2 (+36 days therapy), and T3 (+64 days therapy). The results show that, at diagnosis, ALL children presented with laboratory signs of increased thrombin generation and fibrin formation (i.e. high TAT and D‐dimer levels), fibrinolysis inhibition (i.e. high PAI‐1 level), endothelial activation (i.e., high HMW‐VWF and soluble P‐selectin levels) and inflammation (i.e. high TNF‐alpha and IL‐6 levels). After starting induction therapy, the thrombin generation markers and inflammatory cytokines significantly decreased. To the opposite, PAI‐1 and P‐selectin significantly increased, suggesting an insult by chemotherapy on the vascular endothelium. These effects were more evident during steroid administration. Symptomatic venous thromboembolism (VTE) episodes developed in two cases during induction therapy, which did not allow the evaluation of the predictive value for VTE of laboratory markers. Am. J. Hematol., 2010.

Benefits of prophylaxis versus on-demand treatment in adolescents and adults with severe haemophilia A: the POTTER study

Rigorous evidence is lacking on long-term outcomes of factor VIII (FVIII) prophylaxis initiated in adolescent or adult patients with severe haemophilia A. The prospective, open-label Prophylaxis versus On-demand Therapy Through Economic Report (POTTER) study (ClinicalTrials.gov NCT01159587) compared long-term late secondary prophylaxis (recombinant FVIII-FS 20-30 IU/kg thrice weekly) with on-demand treatment in patients aged 12 to 55 years with severe haemophilia A. The annual number of joint bleeding episodes (primary endpoint), total bleeding episodes, orthopaedic and radiologic (Pettersson) scores, health-related quality of life (HRQoL), pharmacoeconomic impact, and safety were evaluated over a > 5-year period (2004-2010). Fifty-eight patients were enrolled at 11 centres in Italy; 53 (27 prophylaxis, 26 on demand) were evaluated and stratified into 2 age subgroups (12-25 and 26-55 years). Patients receiving prophylaxis experienced a significantly lower number of joint bleeding episodes vs the on-demand group (annualised bleeding rate, 1.97 vs 16.80 and 2.46 vs 16.71 in younger and older patients, respectively; p=0.0043). Results were similar for total bleeding episodes. Prophylaxis was associated with significantly fewer target joints (p< 0.001), better orthopaedic (p=0.0019) and Pettersson (p=0.0177) scores, better HRQoL, and fewer days of everyday activities lost (p< 0.0001) but required significantly higher FVIII product consumption. The POTTER study is the first prospective, controlled trial documenting long-term benefits of late secondary prophylaxis in adolescents and adults with severe haemophilia A. The benefits of reduced bleeding frequency, improved joint status, and HRQoL may offset the higher FVIII consumption and costs.

Familial nonrandom inactivation linked to the X inactivation centre in heterozygotes manifesting haemophilia A

A basic tenet of the Lyon hypothesis is that X inactivation occurs randomly with respect to parental origin of the X chromosome. Yet, nonrandom patterns of X inactivation are common – often ascertained in women who manifest recessive X-linked disorders despite being heterozygous for the mutation. Usually, the cause of skewing is cell selection disfavouring one of the cell lineages created by random X inactivation. We have identified a three generation kindred, with three females who have haemophilia A because of extreme skewing of X inactivation. Although they have both normal and mutant factor VIII (FVIII) alleles, only the mutant one is transcribed; and, they share an XIST allele that is never transcribed. The skewing in this case seems to result from an abnormality in the initial choice process, which prevents the chromosome bearing the mutant FVIII allele from being an inactive X.

Conditions associated with infections of indwelling central venous catheters in cancer patients: a summary

The aim of the present annotation was to summarize the clinical situations that are related to the risk of the development of infections of indwelling central venous catheters (CVCs). The specific catheter type (partially implanted), the underlying disease and its management (acute leukaemia), and the patient’s age (paediatrics) are related to more frequent CVC manipulations, and represent the most important risk factors for infection. This risk is clearly reduced by the use of totally implanted CVCs and the proficient training of the individuals performing the CVC maintenance procedures. The CVC insertion technique (percutaneous versus surgical insertion or radiological suite versus operating theatre) does not seem to modify the risk of infection. Finally, there is disagreement as to whether particular therapeutic procedures, such as bone marrow transplant, and the presence of neutropenia are risk factors associated with a higher incidence of CVCrelated infections.

Prospective study of indwelling central venous catheter-related complications in children with Broviac or clampless valved catheters

Purpose To compare two types of central venous catheters (Broviac and valved clampless) for the incidence and severity of catheter-related complications in children. Patients and Methods The authors report data on the mechanical and infectious complications collected in a prospective analysis of 92 catheters inserted in 82 children from January 2000 to March 2001. Results Two different devices were inserted: 51 Broviac and 41 clampless valved catheters. During the follow-up of 17,803 catheter-days 52 complications were observed: 40 mechanical episodes and 12 infectious events. In the Broviac group the median follow-up was 179 days and the total number of catheter-days was 10,911. A total of 29 complications were observed, occurring in 22 catheters (43%), with an overall incidence of 0.27/100 catheter-days. In the clampless group the median follow-up was 134 days and the total number of catheter days was 6893. A total of 23 complications were observed, occurring in 19 devices (46%), with an incidence of 0.32/100 catheter days. Conclusions There were no major differences in the incidence of mechanical or infectious complications between the two devices. Malfunction was more frequent in Broviac catheters, whereas catheter displacement occurred more frequently in clampless valved catheters. These results show the importance of central venous catheter-related mechanical complications in the management of children with hematologic or oncologic malignancies.

Mechanical complications related to indwelling central venous catheter in pediatric hematology/oncology patients

Indwelling central venous catheters (CVC) are essential devices in the management of children with oncologic/hematologic diseases or following bone marrow transplantation. The authors report data on the mechanical complications observed in pediatric hematology/oncology patients, collected by a retrospective analysis of clinical records of 482 patients in whom 567 indwelling central venous catheters had been inserted from January 1992 to December 1998 at the G. Gaslini Institute. During the study period, 52 episodes of mechanical complications (9%) were observed: mechanical obstruction (24 episodes), catheter dislocation (13), problems related to catheter material (12), and accidental removal (3). In 25 cases removal and replacement of CVC was necessary for the treatment of complications, while medical treatment (thrombolytic-antithrombotic) was successful and well tolerated in 8. The study shows the importance of mechanical complications in children with indwelling CVC for hematologic or oncologic diseases. Moreover, the experience of administering a systemic low-dosage thrombolytic therapy demonstrates new prospects of reducing CVC replacement by restoring CVC viability.

Management and investigation of neonatal thromboembolic events: Genetic and acquired risk factors

Newborns comprise the largest group of children developing thromoboembolic events (TE(S)), due to the peculiarities of their developmental hemostatic system. Moreover, in the sick newborn, especially preterm, numerous acquired perinatal and iatrogenic conditions might result in a disturbance between coagulation and fibrinolysis, leading to thrombus formation. Nevertheless, the contribution of acquired prothrombotic disorders in the pathogenesis of thromboembolic disease in newborns remains poorly defined. Few data are currently available regarding the influence of maternal or fetal genes on thrombotic risk in the fetus and neonate. Ongoing National and International registries are partially answering these questions. The purpose of this review is to evaluate the current knowledge about the role of inherited, acquired perinatal and maternal prothrombotic risk factors in neonatal cerebral nervous system (CNS) thrombotic events and non-CNS thrombotic events.

MLPA assay in F8 gene mutation screening

Besides intron 22 inversion with a reported prevalence of 40–50% in patients with severe haemophilia A (HA) [1], and intron 1 inversion with a reported prevalence of about 2–5% [2], the gene alterations causing HA are spread throughout the gene [3]. Large deletions of one or more gene exons account for about 5% of all cases of severe HA [3]. Over the last few years, we characterized the mutation in a series of patients with severe HA, and traditional PCR failed to amplify one or more exons in four of the 126 patients. As mRNA was not available, we decided to search for further validation. Recently, multiplex ligation-dependent probe amplification (MLPA) has been broadly applied to gene mutation screening to detect exon deletions and duplications [4]. In an ongoing effort to make the technique easier to perform, modifications were made and new MLPA procedures were designed, including the one which contains probes for each of the 26 exons of the F8 gene (SALSA P178 FVIII MLPA kit; MRCAmsterdam, The Netherlands). MLPA evaluation of the multiple amplification relies on the intensity ratio of sample to control, i.e. the relative quantity of each of the PCR products is proportional to the number of copies of the target sequence. Therefore, a ratio proximal or equal to zero indicates deletions of exon/s, a ratio close to 0.5 identifies the presence of the deletion in the heterozygous state. A ratio close to 1.5 identifies the duplication of the exon/s. We present results from a retrospective study that was carried out on patients in whom a deletion or duplication had previously been hypothesized on the basis of PCR alone. MLPA was also applied to the definition of carrier status in the female relatives of the same patients. In fact, the main obstacle to their counselling is the impossibility to demonstrate a heterozygous status because of the amplification of the exon/s that is/are present on the normal X chromosome. Real-time PCR offers the option of carrying out quantification of the amplified material, but simultaneous detection of the gene copy number requires critical consideration of the standardization and reference-curve protocols. In our retrospective study, we demonstrated that carrier status determination via MLPA is straightforward and correct. Multiplex ligation-dependent probe amplification product was performed according to the instructions of the manufacturer. In more detail, 100– 150 ng of genomic DNA were denatured and hybridized with SALSA MLPA probes (60 C overnight). After ligation at 54 C for 15¢, the samples were amplified using a Biometra T personal Thermocycler (Biometra, Göttingen, Germany). One and a half microlitre of PCR products was mixed with 0.3 lL of ROX-500 labelled internal size standard, separated by capillary electrophoresis on a Genetic Analyser 3130 (Applied Biosystem, Foster City, CA, USA), and analysed using the genemapper software (Applied Biosystem). DNA dosage was estimated using coffalyser software (version 6; MRC, Amsterdam, The Netherlands). In the presence of values suggesting deletion/insertion(s) or ambiguous values, MLPA analysis was repeated on independent samples.