Long-term follow-up of liver-directed, adeno-associated vector-mediated gene therapy in the canine model of hemophilia A.

Questions remain concerning the long-term efficacy, safety, and site(s) of transgene expression following adeno-associated vector (AAV) therapy. We report a long-term follow-up of 8 (male = 4, hemizygous, and female = 4, homozygous) dogs with severe hemophilia A treated with a single portal vein infusion of a B-domain-deleted (BDD)-canine FVIII (cFVIII) AAV vector (median dose = 1.25 × 1013 vg/kg, AAV2 = 4, AAV6 = 3, and AAV8 = 1). After a median follow-up of 10.8 years (8.2-12.0 years), persistent FVIII:C (median one-stage = 12.7%, chromogenic = 7.2%) was seen in all responding dogs (n = 6), with improvement in annualized bleed rates (pre = 3.9 vs post = 0.3 event per year; P = .003). Anti-AAV capsid neutralizing antibodies (nAbs) toward the dosed capsid were detected throughout the study, with limited cross-reactivity to other capsids. nAb titers for all capsid serotypes declined with time, although they remained at levels precluding redosing with the same capsid. AAV-BDD-cFVIII DNA was detected in the liver of all dogs (median = 0.15 vg per diploid genome), with lower levels in the spleen in 4 dogs (median = 0.005 vg per diploid genome). Consistent with the liver-specific promoter, BDD-cFVIII mRNA was only detected in the liver. Postmortem examination demonstrated no evidence of chronic liver disease or liver malignancy. Persistent FVIII expression and an improved bleeding phenotype was seen for more than a decade after vector delivery. This is the longest follow-up reported in a preclinical model supporting long-term efficacy and safety of AAV-mediated gene therapy.

Genetic regulation of plasma von Willebrand factor levels in health and disease.

Plasma levels of the multimeric glycoprotein von Willebrand factor (VWF) constitute a complex quantitative trait with a continuous distribution and wide range in the normal population (50-200%). Quantitative deficiencies of VWF (< 50%) are associated with an increased risk of bleeding, whereas high plasma levels of VWF (> 150%) influence the risk of arterial and venous thromboembolism. Although environmental factors can strongly influence plasma VWF levels, it is estimated that approximately 65% of this variability is heritable. Interestingly, although variability in VWF can account for ~ 5% of the genetic influence on plasma VWF levels, other genetic loci also strongly modify plasma VWF levels. The identification of the additional sources of VWF heritability has been the focus of recent observational trait-mapping studies, including genome-wide association studies or linkage analyses, as well as hypothesis-driven research studies. Quantitative trait loci influencing VWF glycosylation, secretion and clearance have been associated with plasma VWF antigen levels in normal individuals, and may contribute to quantitative VWF abnormalities in patients with a thrombotic tendency or type 1 von Willebrand disease (VWD). The identification of genetic modifiers of plasma VWF levels may allow for better molecular diagnosis of type 1 VWD, and enable the identification of individuals at increased risk for thrombosis. Validation of trait-mapping studies with in vitro and in vivo methodologies has led to novel insights into the life cycle of VWF and the pathogenesis of quantitative VWF abnormalities.

The Jamaican Haemophilia Registry: Describing the burden of disease.

INTRODUCTION: Jamaica has an estimated 200 persons with haemophilia (PWH), who face significant constraints in access to specialized haemophilia care, including access to clotting factor concentrates. AIM: The aim of this paper is to establish the current burden of disease in PWH in Jamaica. METHODS: PWH were enrolled through the University Hospital of the West Indies, Jamaica. The impact of haemophilia was assessed using a comprehensive battery of heath outcome measures that included the following: laboratory, clinical information and validated outcome measures of joint structure and function, activity, and health-related quality of life (HRQoL) to provide a health profile of the Jamaican haemophilia population. RESULTS: In all, 45 PWH were registered (mean age: 29, range: 0.17-69 years), including 13 children (<18 years of age) and 32 adults. In this sample, 41 had haemophilia A (30 severe) and 4 had haemophilia B (3 severe); 10 patients with haemophilia A were inhibitor positive. The results indicate that adults with haemophilia in Jamaica have significant joint damage: mean Haemophilia Joint Health Score (HJHS) = 42.1 (SD = 17.3); moderate activity levels - mean Haemophilia Activities List (HAL) score = 64.8 (SD = 17.8); and low HRQoL scores - mean Haemo-QoL-A score = 62.3 (SD = 19.4). Results for children are also reported but should be interpreted with caution due to the small sample size. CONCLUSIONS: There is a very high burden of disease in PWH in Jamaica. The health profiles reported in this paper are an essential first step in advocating for a multidisciplinary Comprehensive Care Program for assessment and care of PWH in Jamaica.

Early cellular interactions and immune transcriptome profiles in human factor VIII-exposed hemophilia A mice.

Essentials Initial immune cell interactions leading to factor (F) VIII immunity are not well characterized. We assessed cellular interactions and expression profiles in hemophilia A mice. MARCO+, followed by SIGLEC1+ and SIGNR1+ macrophages co-localize most with human FVIII. The splenic transcriptome highlights potential therapeutic targets to prevent inhibitors. SUMMARY: Background Developing factor VIII (FVIII) inhibitory antibodies is the most serious complication in hemophilia A treatment, representing a significant health and economic burden. A better understanding of the early events in an immune response leading to this outcome may provide insight into inhibitor development. Objective To identify early mediators of FVIII immunity and to detail immune expression profiles in the spleen and liver. Methods C57Bl/6 F8 E16 knockout mice were infused with 5-20 µg (2000-8000 IU kg-1 ) of recombinant FVIII. Spleens were frozen at various time-points post-infusion and stained for FVIII and cellular markers. Splenic and liver RNA expression analysis was performed 3 h post-infusion of 0.6 µg (240 IU kg-1 ) FVIII by nCounter technology using a 561-gene immunology panel. Results FVIII localization in the spleen did not change over 2.5 h. We observed significantly higher co-localization of FVIII with MARCO+ cells compared with SIGLEC1+ and SIGNR1+ in the splenic marginal zone. FVIII exhibited little co-localization with CD11c+ dendritic cells and the macrophage mannose receptor, CD206. Following FVIII infusion, the splenic mRNA profiling identified genes such as Tnfaip6 and Il23r, which are implicated in chemotaxis and a proinflammatory Th17 response, respectively. In contrast, an upregulation of Gfi1 in the liver suggests an anti-inflammatory environment. Conclusions FVIII co-localizes predominantly with marginal zone macrophages (MARCO+ ) in the murine spleen following intravenous infusion. Targeting pathways that are implicated in the early FVIII innate immune response in the spleen may lead to therapeutic interventions to prevent inhibitor formation.

Abnormal von Willebrand factor secretion, factor VIII stabilization and thrombus dynamics in type 2N von Willebrand disease mice.

Essentials Type 2N von Willebrand disease involves impaired von Willebrand factor to factor VIII binding. Type 2N von Willebrand disease mutations exhibit qualitative and mild quantitative deficiencies. Type 2N von Willebrand disease mice exhibit unstable venous hemostatic thrombi. The factor VIII-binding ability of von Willebrand factor regulates arteriole thrombosis dynamics. SUMMARY: Background von Willebrand factor (VWF) and factor VIII (FVIII) circulate as a non-covalent complex, with VWF serving as the carrier for FVIII. VWF indirectly influences secondary hemostasis by stabilizing FVIII and transporting it to the site of primary hemostasis. Type 2N von Willebrand disease involves impaired binding of VWF to FVIII, resulting in decreased plasma levels of FVIII. Objectives In these studies, we characterize the impact of three type 2N VWD variants (R763A, R854Q, R816W) on VWF secretion, FVIII stabilization and thrombus formation in a murine model. Methods Type 2N VWD mice were generated by hydrodynamic injections of mutant murine VWF cDNAs and the influence of these variants on VWF secretion and FVIII binding was evaluated. In vivo hemostasis and the dynamics of thrombus formation and embolization were assessed using a murine tail vein transection hemostasis model and an intravital thrombosis model in the cremaster arterioles. Results Type 2N VWD variants were associated with decreased VWF secretion using cell and animal-based models. FVIII-binding to type 2N variants was impaired in vitro and was variably stabilized in vivo by expressed or infused 2N variant VWF protein. Both transgenic type 2N VWD and FVIII knockout (KO) mice demonstrated impaired thrombus formation associated with decreased thrombus stability. Conclusions The type 2N VWD phenotype can be recapitulated in a murine model and is associated with both quantitative and qualitative VWF deficiencies and impaired thrombus formation. Patients with type 2N VWD may have normal primary hemostasis formation but decreased thrombus stability related to ineffective secondary hemostasis.

Discrepant platelet and plasma von Willebrand factor in von Willebrand disease patients with p.Pro2808Leufs*24.

Essentials von Willebrand factor (VWF) is synthesized in endothelial cells and platelet precursors. Type 3 patients with Pro2808Leufs*24 have lower bleeding scores than other type 3s. The Pro2808Leufs*24 variant was examined in patient platelets and endothelial cells. Type 3s with this variant contain releaseable VWF, possibly reducing bleeding. SUMMARY: Background A novel variant, p.Pro2808Leufs*24, in the von Willebrand factor (VWF) gene was previously identified in the Canadian von Willebrand disease (VWD) patient population. Clinical observations of type 3 VWD patients with this variant indicate a milder bleeding phenotype compared with other type 3 patients. Objective To assess the effect of the Pro2808Leufs*24 variant on the molecular pathogenesis of VWD and correlate this with the phenotype observed in patients. Patients/Methods Phenotypic data from individuals in the Canadian type 3 VWD study were analyzed. VWF expression in platelets and plasma was assessed via immunoblotting. Cellular expression of VWF in platelets and blood outgrowth endothelial cells (BOEC) was examined via immunofluorescence microscopy and biochemical analysis in a type 3 index case and family member with Pro2808Leufs*24. Results Twenty-six individuals with the Pro2808Leufs*24 variant (16 type 3 VWD homozygous or compound heterozygous and 10 heterozygous family members) were studied. Bleeding scores were lower in type 3 patients with Pro2808Leufs*24 compared with type 3 patients with other variants, confirming a milder bleeding phenotype. Immunoblotting of platelet lysates detected VWF in the platelets of type 3 patients with Pro2808Leufs*24. Examination of an index case detected VWF within platelets via immunofluorescence microscopy, and in vitro experiments showed that this VWF was released upon platelet activation. Patient BOECs showed decreased VWF synthesis and secretion, although some VWF-containing granules were observed. Conclusion Type 3 VWD patients with the Pro2808Leufs*24 have bioavailable platelet-derived VWF that may produce a milder bleeding phenotype than other type 3s.

Factor VIII inhibitors: Advances in basic and translational science.

In the treatment of hemophilia A, the 20%-30% risk of developing of anti-factor VIII (FVIII) antibodies, or inhibitors, is the dominant concern among healthcare providers. Immune tolerance induction remains the only effective method of eradicating inhibitors in approximately 75% of patients, but is accompanied by significant emotional and economical burden. While certain risk factors, such as the type of FVIII mutation, offer some insight, there remains no strategy to confidently predict the development of an inhibitor. Moreover, even if such a predictive tool existed, there is currently no proven protocol for tolerance induction of a previously untreated patient. In recent years, the growing body of knowledge concerning the fundamental immunology of inhibitors has shed light on potential therapeutic interventions. In this review, we highlight these new findings and their influences on translational medicine.

Histones link inflammation and thrombosis through the induction of Weibel-Palade body exocytosis.

Essentials Dysregulated DNA and histone release can promote pathological immunothrombosis. Weibel-Palade bodies (WPBs) are sentinel-like organelles that respond to proinflammatory stimuli. Histones induce WPB exocytosis in a caspase, calcium and charge-dependent mechanism. A targetable axis may exist between DNA/histones and WPBs in inflammation and immunothrombosis. SUMMARY: Background Damage-associated molecular patterns (DAMPs), including molecules such as DNA and histones, are released into the blood following cell death. DAMPs promote a procoagulant phenotype through enhancement of thrombin generation and platelet activation, thereby contributing to immunothrombosis. Weibel-Palade bodies (WPBs) are dynamic endothelial cell organelles that contain procoagulant and proinflammatory mediators, such as von Willebrand factor (VWF), and are released in response to cell stresses. VWF mediates platelet adhesion and aggregation, and has been implicated as a procoagulant component of the innate immune response. Objective To determine the influence of histones and DNA on WPB release, and characterize their association in models of inflammation. Methods We treated C57BL/6J mice and cultured endothelial cells with histones (unfractionated, lysine-rich or arginine-rich) and DNA, and measured WPB exocytosis. We used inhibitors to determine a mechanism of histone-induced WPB release in vitro. We characterized the release of DAMPs and WPBs in response to acute and chronic inflammation in human and murine models. Results and conclusions Histones, but not DNA, induced the release of VWF (1.46-fold) from WBPs and caused thrombocytopenia (0.74-fold), which impaired arterial thrombus formation in mice. Histones induced WPB release from endothelial cells in a caspase-dependent, calcium-dependent and charge-dependent manner, and promoted platelet capture in a flow chamber model of VWF-platelet string formation. The levels of DAMPs and WPB-released proteins were elevated during inflammation, and were positively correlated in chronic inflammation. These studies showed that DAMPs can regulate the function and level of VWF by inducing its release from endothelial WPBs. This DAMP-WPB axis may propagate immunothrombosis associated with inflammation.

Recommendations for authors of manuscripts reporting inhibitor cases developed in previously treated patients with hemophilia: communication from the SSC of the ISTH.

Aim The scope of this recommendation is to provide guidance for reporting of inhibitor cases in previously treated patients (PTPs) with hemophilia A. This guidance is intended to improve transparency and completeness of reporting of observed events; it does not cover planning, executing or analyzing original studies aimed at the assessment of inhibitor rates. Recommendation We recommend that for each case of inhibitor development reported in a published paper, a paragraph or a table is included in the main publication reporting as a minimum the underlined data fields in Table . We recommend transparent reporting when any of the suggested information is not available. We recommend that particular care is used in reporting the timeline of events by clearly identifying a reference time-point. We suggest that journals in the field adopt this guidance as instructions for the authors and as a guide for reviewers. Conclusion Development of inhibitors in PTPs is a very rare event. Standardized reporting of inhibitor characteristics will contribute to generating a body of evidence otherwise not available. Case by case reporting of the recommended data elements may shed light on the natural history and risk factors of inhibitor development in PTPs and be useful for tailoring care in similar future cases.

Alternative therapies for the management of inhibitors.

The development of inhibitors to factor VIII (FVIII) or factor IX (FIX) remains a major treatment complication encountered in the treatment of haemophilia. Not all patients with even the same severity and genotype develop inhibitors suggesting an underlying mechanism of tolerance against FVIII- or FIX-related immunity. One mechanism may be central tolerance observed in patients in whom the FVIII mutation enables some production of the protein. The other is a peripheral tolerance mechanism which may be evident in patients with null mutation. Recently, recombinant porcine FVIII (rpFVIII, Obixur, OBI-1, BAX801) has been developed for the haemostatic treatment of both congenital haemophilia with inhibitor (CHAWI) and acquired haemophilia A (AHA). In 28 subjects with AHA with life-/limb-threatening bleeding, rpFVIII reduced or stopped bleeding in all patients within 24 h. The cross-reactivity of anti-human FVIII antibodies to rpFVIII remains around 30-50%. Recently, new therapeutics based on the quite novel concepts have been developed and clinical studies are ongoing. These are humanized asymmetric antibody mimicking FVIIIa function by maintaining a suitable interaction between FIXa and FX (Emicizumab, ACE910), and small interfering RNAs (siRNA, ALN-AT3) suppress liver production of AT through post-transcriptional gene silencing and a humanized anti-TFPI monoclonal antibody (Concizumab). Their main advantages are longer half-life, subcutaneous applicability and efficacy irrespective of the presence of inhibitors which will make it easier to initiate more effective treatment especially early childhood.

Canine models of inherited bleeding disorders in the development of coagulation assays, novel protein replacement and gene therapies.

Animal models of inherited bleeding disorders are important for understanding disease pathophysiology and are required for preclinical assessment of safety prior to testing of novel therapeutics in human and veterinary medicine. Experiments in these animals represent important translational research aimed at developing safer and better treatments, such as plasma-derived and recombinant protein replacement therapies, gene therapies and immune tolerance protocols for antidrug inhibitory antibodies. Ideally, testing is done in animals with the analogous human disease to provide essential safety information, estimates of the correct starting dose and dose response (pharmacokinetics) and measures of efficacy (pharmacodynamics) that guide the design of human trials. For nearly seven decades, canine models of hemophilia, von Willebrand disease and other inherited bleeding disorders have not only informed our understanding of the natural history and pathophysiology of these disorders but also guided the development of novel therapeutics for use in humans and dogs. This has been especially important for the development of gene therapy, in which unique toxicities such as insertional mutagenesis, germ line gene transfer and viral toxicities must be assessed. There are several issues regarding comparative medicine in these species that have a bearing on these studies, including immune reactions to xenoproteins, varied metabolism or clearance of wild-type and modified proteins, and unique tissue tropism of viral vectors. This review focuses on the results of studies that have been performed in dogs with inherited bleeding disorders that closely mirror the human condition to develop safe and effective protein and gene-based therapies that benefit both species.

Aging and ABO blood type influence von Willebrand factor and factor VIII levels through interrelated mechanisms.

UNLABELLED: Essentials von Willebrand factor (VWF) and factor VIII (FVIII) levels are modulated by age and ABO status. The effect of aging and ABO blood type on VWF and FVIII was assessed in 207 normal individuals. Aging and ABO blood type showed combined and bidirectional influences on VWF and FVIII levels. Aging and ABO blood type influence VWF levels through both secretion and clearance mechanisms. SUMMARY: Background The effect of aging and ABO blood type on plasma levels of von Willebrand factor (VWF) and factor VIII (FVIII) have been widely reported; however, a comprehensive analysis of their combined effect has not been performed and the mechanisms responsible for the age-related changes have not been determined. Objectives To assess the influence of aging and ABO blood type on VWF and FVIII levels, and to evaluate the contribution of VWF secretion and clearance to the age-related changes. Methods A cross-sectional observational study was performed in a cohort of 207 normal individuals, whose levels of VWF, FVIII, VWF propeptide (VWFpp), VWFpp/VWF:Ag ratio and blood type A antigen content on VWF (A-VWF) were quantified. Results Aging and ABO blood type exerted interrelated effects on VWF and FVIII plasma levels, because the age-related increase in both proteins was significantly higher in type non-O individuals (ß = 0.011 vs. 0.005). This increase with age in non-O subjects drove the differences between blood types in VWF levels, as the mean difference increased from 0.13 U/mL in the young to 0.57 U/mL in the old. Moreover, A-VWF was associated with both VWF antigen (ß = 0.29; 95% confidence interval [CI], 0.09, 0.50) and VWF clearance (ß = -0.15; 95% CI, -0.25, -0.06). We also documented an effect of ABO blood type on VWF secretion with aging, as old individuals with blood type non-O showed higher levels of VWFpp (mean difference 0.29 U/mL). Conclusions Aging and ABO blood type have an interrelated effect on VWF and FVIII levels, where the effect of one is significantly influenced by the presence of the other.

Porcine recombinant factor VIII (Obizur; OBI-1; BAX801): product characteristics and preclinical profile.

INTRODUCTION: Acquired haemophilia A (AHA) is a rare, often severe, auto-immune bleeding disorder caused by the development of inhibitory antibodies (inhibitors) to factor VIII (FVIII). Bypassing agents, recombinant activated FVII or activated prothrombin complex concentrate, are currently recommended as first-line treatments to control bleeding events in patients with AHA. AIM: A plasma-derived porcine FVIII (Hyate:C, Ipsen, UK) was used as a first-line treatment for AHA but was discontinued in 2004 due to viral safety concerns. A recombinant pFVIII (rpFVIII), Obizur (OBI-1; BAX801), which is expected to have a similar efficacy profile to Hyate:C but with a superior safety profile was developed and recently approved by the US Food and Drug Administration for the treatment of AHA. METHODS: Obizur manufacturing begins with the expression of B domain deleted rpFVIII by genetically modified baby hamster kidney-derived cells. The final purified and lyophilized drug product has a negligible risk of viral contamination and contains no animal-derived plasma proteins. Obizur was evaluated for immunogenicity, tolerability, pharmacokinetics and bleeding times in preclinical models including in haemophiliac dogs, cynomolgus monkeys and FVIII-knockout mice. RESULTS: Preclinical animal studies show that the efficacy and immunogenicity of Obizur are similar to that of Hyate:C and that Obizur has a more favourable safety profile. CONCLUSIONS: Obizur is a highly purified recombinant porcine FVIII drug product that has been demonstrated to have a favourable safety and efficacy profile when compared with Hyate:C and can be a valuable treatment option for control of bleeding in AHA patients.

Changes in von Willebrand factor level and von Willebrand activity with age in type 1 von Willebrand disease.

In a normal population, VWF plasma levels (VWF:Ag) and VWF activity (VWF:RCo) increase by approximately 0.17 and 0.15 IU mL(-1) per decade, but the influence of age is unknown in patients with type 1 von Willebrand disease (VWD). In a retrospective cohort study, the medical records of 31 type 1 VWD patients over the age of 30, who had been followed for ≥5 years, were reviewed for baseline clinical data and previously performed VWF:Ag, VWF:RCo and factor VIII levels ( FVIII: C). VWF multimer analysis was normal in 28/31 cases performed. Mean age at diagnosis was 33 (range 16-60 years), and duration of follow-up ranged from 5 to 26 years (mean 11 years). Patients had 2-10 time points of VWD testing (mean of 5.2). The mean VWF:Ag, VWF:RCo and FVIII: C at time of diagnosis were 0.44 IU mL(-1) 0.34 IU mL(-1) and 0.75 IU mL(-1) . At last follow-up, the mean VWF:Ag, VWF:RCo and FVIII: C were significantly increased to 0.71 IU L(-1) , 0.56 IU mL(-1) and 0.90 IU mL(-1) (P ≤ 0.001, <0.001, and 0.0081 respectively). Here 18/31 patients had VWF:Ag, VWF:RCo and FVIII: C levels that increased into the normal range. The rate of change in VWF:Ag, VWF:RCo and FVIII was 0.30 IU mL(-1) (0.21-0.39, CI 95%, P < 0.0001), 0.20 IU mL(-1) per decade (0.13-0.27, CI 95%, P = 0.0001) and 0.20 IU mL(-1) (0.11-0.29, CI 95%, P = 0.0011). Patients with type 1 VWD experience age-related increases to VWF:Ag and VWF:RCo which can result in normalization of VWF levels. Further studies are required to determine if the bleeding phenotype resolves with the increases in VWF:Ag and VWF:RCo levels.

Recombinant factor VIII Fc fusion protein: extended-interval dosing maintains low bleeding rates and correlates with von Willebrand factor levels.

BACKGROUND: Routine prophylaxis with replacement factor VIII (FVIII) - the standard of care for severe hemophilia A - often requires frequent intravenous infusions (three or four times weekly). An FVIII molecule with an extended half-life could reduce infusion frequency. The A-LONG study established the safety, efficacy and prolonged pharmacokinetics of recombinant FVIII Fc fusion protein (rFVIIIFc) in previously treated adolescents and adults with severe hemophilia A. OBJECTIVE: In this post hoc analysis, we investigated the relationship between subjects' prestudy (FVIII) and on-study (rFVIIIFc) regimens. METHODS: We analyzed two subgroups of subjects: prior prophylaxis and on-study individualized prophylaxis (n = 80), and prior episodic treatment and on-study weekly prophylaxis (n = 16). Subjects' prestudy dosing regimens and bleeding rates were compared with their final rFVIIIFc regimens and annualized bleeding rates (ABRs) in the last 3 months on-study. Dosing regimen simulations based on population pharmacokinetics models for rFVIII and rFVIIIFc were performed. RESULTS: As compared with their prestudy regimen, 79 of 80 (98.8%) subjects on individualized rFVIIIFc prophylaxis decreased their infusion frequency. Overall ABRs were low, with comparable factor consumption. Longer dosing intervals, including 5-day dosing, were associated with higher baseline von Willebrand factor antigen levels. Simulated dosing regimens predicted a greater proportion of subjects with steady-state FVIII activity trough levels of ≥ 1 IU dL(-1) (1%) with rFVIIIFc than with equivalent rFVIII regimens. CONCLUSION: These results suggest that patients on rFVIIIFc prophylaxis can reduce their infusion frequency as compared with their prior FVIII regimen while maintaining low bleeding rates, affording more patients trough levels of ≥ 1 IU dL(-1) than with rFVIII products requiring more frequent dosing regimens.

Incidence and risk factors for inhibitor development in previously untreated severe haemophilia A patients born between 2005 and 2010.

The objective of this study was to evaluate the inhibitor development (ID) in previously untreated patients (PUPs) with severe haemophilia A (FVIII ≤ 0.01 IU mL(-1) ). All Canadian Haemophilia Treatment Centres completed a questionnaire on patients born between September 2005 and August 2010 and followed for up to 7 years. Eligible patients had at least 20 exposure days (ED) or had developed an inhibitor. The odds ratio (OR) and 95% confidence intervals (95% CI) for risk factors to develop an inhibitor were estimated using unconditional logistic regression. A total of 99 haemophilia A PUPs were studied. Thirty-four (34%) developed an inhibitor (24/34 of high titre). Inhibitors developed in 25/63 (40%) patients with a high-risk mutation. ID was most frequent in Aboriginals (86%). Dose intensity (IU kg(-1)  day(-1) X number of ED) at first exposure to factor VIII (FVIII) was associated with a crude OR increase of 1.10 (95% CI: 0.99-1.23) with each increase of 100 dose-intensity units. Haemarthrosis and intracranial bleeding as the indication for first exposure to FVIII concentrate were associated with a crude OR for ID of 7.63 (95% CI: 2.14-27.17) and 5.08 (95% CI: 1.11-23.31) respectively. ID according to FVIII concentrate used was: Advate (®) 18/50 (36%), Kogenate FS(®) or Helixate FS(®) 15/36 (42%), Wilate(®) 0/11 and Xyntha(®) 1/2. In multivariate analysis, Aboriginal ethnicity (OR = 11.69; 95% CI: 1.11-122.86) and haemarthrosis (OR = 4.49; 95% CI: 1.08-18.61) were statistically significant. The cumulative incidence of ID in severe haemophilia A PUPs was 34% and varied according to ethnicity, type of bleeding at first ED, type of FVIII product and dose intensity at first exposure.

First analysis of 10-year trends in national factor concentrates usage in haemophilia: data from CHARMS, the Canadian Hemophilia Assessment and Resource Management System.

The Canadian Hemophilia Assessment and Resource Management System (CHARMS) tracks factor concentrates (FC) from the sole suppliers, Canadian Blood Services (CBS) and Hema-Quebec (HQ), to hospitals and to patients' homes. Patients FC infusion data are entered into CHARMS at Canadian Hemophilia Treatment Centres (HTCs) then exported to the national database (CentrePoint). From 2000 to 2009, 2260 registered haemophilia A or B patients received FVIII (1,009,097,765 IU) and FIX (272,406,859 IU). Over 91% of FVIII and over 84% of FIX was infused at home. Utilization of FVIII progressively increased; this was accounted for by an increase in the number of patients treated (r = 0.97; P < 0.001), there being a linear relationship between the increase in utilization and the increase in number of patients treated (P < 0.001). There was also a correlation with the annual amount used per patient (r = 0.95; P < 0.001). Utilization of FIX did not increase over time. The highest proportional utilization of both FVIII and FIX was for prophylaxis, and this proportion progressively increased being, in year 10 (2009), 77% and 66% for FVIII and FIX respectively. The proportion used for bleeding remained steady; in year 10 that proportion was 14% for FVIII and 26% for FIX, the use per patient for bleeding decreasing. The HTC-based CHARMS tracking system is essential, in Canada, for analysing indications for infusion, for predicting utilization and planning for future needs.