Clinical and economic burden of immune tolerance induction in entire patients with hemophilia A: Insights from a real-world Korean setting.

INTRODUCTION: The most notable challenge facing hemophilia A treatment is the development of inhibitors against factor VIII, resulting in increased clinical and socioeconomic burdens due to the need for expensive bypassing agents (BPAs). Although immune tolerance induction (ITI) is currently the primary approach for inhibiting and reducing the inhibitors, the lengthy duration of ITI necessitates the continued use of BPA to manage bleeding episodes. In this study, we aimed to obtain real-world evidence on the clinical and economic aspects and associated burdens experienced by patients with hemophilia A with inhibitors undergoing ITI in Korea. METHODS: Claims data from January 1, 2007, to December 31, 2020, were used in this study. The study cohort comprised patients with hemophilia A undergoing ITI, who were categorized into three groups: successful, failed, or continuation of ITI. We evaluated clinical and economic burdens, including monthly healthcare visits, medication costs, and total medical expenses. RESULTS: The study involved 33 cases of ITI across 32 patients. Excluding seven continuation cases where success could not be determined at the observation point, the estimated success rate of ITI was 80.8 %. The median duration of ITI for all patients was 25.7 months. While no significant disparities were noted in the ITI duration between successful and unsuccessful cases (24.51 vs. 25.66 months), substantial discrepancies were observed in the duration of BPA usage (11.10 vs. 25.66 months) and the number of prescribed BPAs (1.79 vs. 2.97). CONCLUSION: Successful ITI reduced both clinical and economic burdens, resulting in decreased monthly medication expenses and overall medical costs.

Factor VIII moiety of recombinant Factor VIII Fc fusion protein impacts Fc effector function and CD16+ NK cell activation.

Recombinant Factor VIII-Fc fusion protein (rFVIIIFc) is an enhanced half-life therapeutic protein product used for the management of hemophilia A. Recent studies have demonstrated that rFVIIIFc interacts with Fc gamma receptors (FcγR) resulting in the activation or inhibition of various FcγR-expressing immune cells. We previously demonstrated that rFVIIIFc, unlike recombinant Factor IX-Fc (rFIXFc), activates natural killer (NK) cells via Fc-mediated interactions with FcγRIIIA (CD16). Additionally, we showed that rFVIIIFc activated CD16+ NK cells to lyse a FVIII-specific B cell clone. Here, we used human NK cell lines and primary NK cells enriched from peripheral blood leukocytes to study the role of the FVIII moiety in rFVIIIFc-mediated NK cell activation. Following overnight incubation of NK cells with rFVIIIFc, cellular activation was assessed by measuring secretion of the inflammatory cytokine IFNγ by ELISA or by cellular degranulation. We show that anti-FVIII, anti-Fc, and anti-CD16 all inhibited indicating that these molecules were involved in rFVIIIFc-mediated NK cell activation. To define which domains of FVIII were involved, we used antibodies that are FVIII domain-specific and demonstrated that blocking FVIII C1 or C2 domain-mediated membrane binding potently inhibited rFVIIIFc-mediated CD16+ NK cell activation, while targeting the FVIII heavy chain domains did not. We also show that rFVIIIFc binds CD16 with about five-fold higher affinity than rFIXFc. Based on our results we propose that FVIII light chain-mediated membrane binding results in tethering of the fusion protein to the cell surface, and this, together with increased binding affinity for CD16, allows for Fc-CD16 interactions to proceed, resulting in NK cellular activation. Our working model may explain our previous results where we observed that rFVIIIFc activated NK cells via CD16, whereas rFIXFc did not despite having identical IgG1 Fc domains.

Race and ethnicity and the success of immune tolerance induction among people with severe haemophilia A in the United States.

INTRODUCTION: Immune tolerance induction (ITI) is the only treatment to eradicate inhibitors in people with severe haemophilia A with inhibitors. Since the risk of inhibitor development is greater among Black and Hispanic persons, it has been hypothesized that race and ethnicity may influence ITI success. Limited studies have evaluated this hypothesis. AIM: To examine the success of ITI according to race and ethnicity. METHODS: Participants who entered the Community Counts (CC) Registry between 2013 and 2017, were aged ≥3 years at study entry, and received ITI were included (n = 559). The proportion of participants with successful ITI was examined with adjusted prevalence ratios (aPRs) and corresponding 95% confidence intervals (95% CIs). RESULTS: Among 559 participants, 56.9%, 19.1%, 18.1% and 4.3% were Non-Hispanic (NH) White, NH Black, Hispanic and Asian, respectively, and 1.7% were coded as other or missing. Approximately 80% of Hispanic, NH Black and NH White participants had good/very good prognosis, defined as having a pre-ITI peak inhibitor of < 200 Bethesda Units per millilitre. Nearly 60% of participants (59.7%) achieved successful ITI, 20.7% and 19.5% experienced partially successful or failed ITI, respectively. Successful ITI was non-significantly lower in NH Black (54.2%; aPR = 0.95, 95% CI 0.62-1.44) and Hispanic (55.4%; aPR = 0.89, 95% CI 0.71-1.13) relative to NH White participants (62.6%). CONCLUSION: In this study, 60% of participants in the CC Registry had successful ITI, consistent with previous studies. The proportion with successful ITI was generally comparable across racial and ethnic groups with similar prognosis. These findings do not support the hypothesis that ITI response varies according to race or ethnicity.

Multicenter assessment and longitudinal study of the prevalence of antibodies and related adaptive immune responses to AAV in adult males with hemophilia.

Adeno-associated virus (AAV) based gene therapy has demonstrated effective disease control in hemophilia. However, pre-existing immunity from wild-type AAV exposure impacts gene therapy eligibility. The aim of this multicenter epidemiologic study was to determine the prevalence and persistence of preexisting immunity against AAV2, AAV5, and AAV8, in adult participants with hemophilia A or B. Blood samples were collected at baseline and annually for ≤3 years at trial sites in Austria, France, Germany, Italy, Spain, and the United States. At baseline, AAV8, AAV2, and AAV5 neutralizing antibodies (NAbs) were present in 46.9%, 53.1%, and 53.4% of participants, respectively; these values remained stable at Years 1 and 2. Co-prevalence of NAbs to at least two serotypes and all three serotypes was present at baseline for ~40% and 38.2% of participants, respectively. For each serotype, ~10% of participants who tested negative for NAbs at baseline were seropositive at Year 1. At baseline, 38.3% of participants had detectable cell mediated immunity by ELISpot, although no correlations were observed with the humoral response. In conclusion, participants with hemophilia may have significant preexisting immunity to AAV capsids. Insights from this study may assist in understanding capsid-based immunity trends in participants considering AAV vector-based gene therapy.

Age-stratified adeno-associated virus serotype 3 neutralizing and total antibody prevalence in hemophilia A patients from India.

Gene therapy using an adeno-associated virus (AAV) vector offers a new treatment option for individuals with monogenetic disorders. The major bottleneck is the presence of pre-existing anti-AAV antibodies, which impacts its use. Even very low titers of neutralizing antibodies (NAb) to capsids from natural AAV infections have been reported to inhibit the transduction of intravenously administered AAV in animal models and are associated with limited efficacy in human trials. Assessing the level of pre-existing NAb is important for determining the primary eligibility of patients for AAV vector-based gene therapy clinical trials. Techniques used to screen AAV-antibodies include AAV capsid enzyme-linked immunosorbent assay (ELISA) and transduction inhibition assay (TIA) for detecting total capsid-binding (TAb) and Nab, respectively. In this study, we screened 521 individuals with hemophilia A from India for TAb and NAb using ELISA and TIA, respectively. The prevalence of TAb and NAb in hemophilia A patients from India were 96% and 77.5%, respectively. There was a significant increase in anti-AAV3 NAb prevalence with age in the hemophilia A patient group from India. There was a trend in anti-AAV3 TAb positivity between the pediatric age group (94.4%) and the adult age group (97.4%).

Nonhuman glycans can regulate anti-factor VIII antibody formation in mice.

Recombinant factor VIII (FVIII) products represent a life-saving intervention for patients with hemophilia A. However, patients can develop antibodies against FVIII that prevent its function and directly increase morbidity and mortality. The development of anti-FVIII antibodies varies depending on the type of recombinant product used, with previous studies suggesting that second-generation baby hamster kidney (BHK)-derived FVIII products display greater immunogenicity than do third-generation Chinese hamster ovary (CHO)-derived FVIII products. However, the underlying mechanisms responsible for these differences remain incompletely understood. Our results demonstrate that BHK cells express higher levels of the nonhuman carbohydrate α1-3 galactose (αGal) than do CHO cells, suggesting that αGal incorporation onto FVIII may result in anti-αGal antibody recognition that could positively influence the development of anti-FVIII antibodies. Consistent with this, BHK-derived FVIII exhibits increased levels of αGal, which corresponds to increased reactivity with anti-αGal antibodies. Infusion of BHK-derived, but not CHO-derived, FVIII into αGal-knockout mice, which spontaneously generate anti-αGal antibodies, results in significantly higher anti-FVIII antibody formation, suggesting that the increased levels of αGal on BHK-derived FVIII can influence immunogenicity. These results suggest that posttranslational modifications of recombinant FVIII products with nonhuman carbohydrates may influence the development of anti-FVIII antibodies.

Predictors of the outcome of immune tolerance induction in patients with haemophilia A and inhibitors: The Brazilian Immune Tolerance (BrazIT) Study protocol.

The development of inhibitors is the main complication of haemophilia A (HA) treatment. Immune tolerance induction (ITI) is the treatment of choice for inhibitor eradication. We describe the methodology of the Brazilian Immune Tolerance Induction (BrazIT) Study, aimed to identify clinical, genetic, and immune biomarkers associated with response to ITI and inhibitor recurrence. This cohort study includes people with HA (PwHA) and inhibitors (a) who require bypassing agents to treat and/or prevent bleeding, and (b) who are at any stage of ITI treatment. Patients are included in each haemophilia treatment centre (HTC). Factor VIII (FVIII) and inhibitor assessments are performed at local laboratories of each HTC. The ITI regimen followed the national protocol of the Brazilian Ministry of Health. All PwHA starts with low-dose ITI (50 IU/kg three times weekly); high-dose regimen (100 IU/kg daily) is used if there is lack of response to the low-dose ITI. Outcomes are classified as total or partial success, and failure. Standardized case report forms with clinical, laboratory, and treatment data are collected from medical files and interviews. Blood samples are collected for genetic and immune biomarkers at the time of inclusion in the study and at the end of ITI. The study is ongoing and, currently, 202/250 (80.8%) PwHA from 15 HTCs have been included. BrazIT Study is the largest cohort of PwHA and inhibitor under treatment with the same ITI regimen reported to date. This study is likely to contribute with novel predictors of ITI response.

Structure of Blood Coagulation Factor VIII in Complex With an Anti-C2 Domain Non-Classical, Pathogenic Antibody Inhibitor.

Factor VIII (fVIII) is a procoagulant protein that binds to activated factor IX (fIXa) on platelet surfaces to form the intrinsic tenase complex. Due to the high immunogenicity of fVIII, generation of antibody inhibitors is a common occurrence in patients during hemophilia A treatment and spontaneously occurs in acquired hemophilia A patients. Non-classical antibody inhibitors, which block fVIII activation by thrombin and formation of the tenase complex, are the most common anti-C2 domain pathogenic inhibitors in hemophilia A murine models and have been identified in patient plasmas. In this study, we report on the X-ray crystal structure of a B domain-deleted bioengineered fVIII bound to the non-classical antibody inhibitor, G99. While binding to G99 does not disrupt the overall domain architecture of fVIII, the C2 domain undergoes an ~8 Å translocation that is concomitant with breaking multiple domain-domain interactions. Analysis of normalized B-factor values revealed several solvent-exposed loops in the C1 and C2 domains which experience a decrease in thermal motion in the presence of inhibitory antibodies. These results enhance our understanding on the structural nature of binding non-classical inhibitors and provide a structural dynamics-based rationale for cooperativity between anti-C1 and anti-C2 domain inhibitors.

Risk factors for antibody formation in children with hemophilia: methodological aspects and clinical characteristics of the HEMFIL cohort study.

Up to 35% of patients with hemophilia A and 5% with hemophilia B develop neutralizing antibodies which can inhibit the therapeutic activity of factor replacement (inhibitors). Despite the clinical relevance of antifactor VIII and IX neutralizing antibodies, there is still a major gap on the knowledge of risk factors for their development. Furthermore, most of the studies on risk factors for inhibitor development come from Caucasian and Afro-American populations. The HEMFIL is a Brazilian prospective cohort study of previously untreated children with hemophilia, which primary aim is to identify new risk factors related to inhibitor development. This manuscript aims at describing the study design and its methodology. After the diagnosis, children are followed up to 75 exposure days or to inhibitor development. Standardized forms and blood samples are collected to describe clinical characteristics and to perform the measurement of immunological and genetic biomarkers at three time points; Inclusion time (T0), at inhibitor development or at 75 exposure days without inhibitors (T1) and after immune tolerance induction for patients in whom it is indicated and performed (T2). Currently, 120 children have been included, of whom, 95 have completed the follow-up. For severe/moderately severe hemophilia A, the cumulative incidence of inhibitors at 75 exposure days was 35% (95% confidence interval, 26-46%). The inclusion of additional patients and a longer follow-up will allow the analysis of risk factors for inhibitor development.

CAR- and TRuC-redirected regulatory T cells differ in capacity to control adaptive immunity to FVIII.

Regulatory T cells (Tregs) control immune responses in autoimmune disease, transplantation, and enable antigen-specific tolerance induction in protein-replacement therapies. Tregs can exert a broad array of suppressive functions through their T cell receptor (TCR) in a tissue-directed and antigen-specific manner. This capacity can now be harnessed for tolerance induction by "redirecting" polyclonal Tregs to overcome low inherent precursor frequencies and simultaneously augment suppressive functions. With the use of hemophilia A as a model, we sought to engineer antigen-specific Tregs to suppress antibody formation against the soluble therapeutic protein factor (F)VIII in a major histocompatibility complex (MHC)-independent fashion. Surprisingly, high-affinity chimeric antigen receptor (CAR)-Treg engagement induced a robust effector phenotype that was distinct from the activation signature observed for endogenous thymic Tregs, which resulted in the loss of suppressive activity. Targeted mutations in the CD3ζ or CD28 signaling motifs or interleukin (IL)-10 overexpression were not sufficient to restore tolerance. In contrast, complexing TCR-based signaling with single-chain variable fragment (scFv) recognition to generate TCR fusion construct (TRuC)-Tregs delivered controlled antigen-specific signaling via engagement of the entire TCR complex, thereby directing functional suppression of the FVIII-specific antibody response. These data suggest that cellular therapies employing engineered receptor Tregs will require regulation of activation thresholds to maintain optimal suppressive function.

Acquired hemophilia A associated with Epstein-Barr-virus-associated T/natural killer-cell lymphoproliferative disease: A case report.

INTRODUCTION: Acquired hemophilia A (AHA) is a rare bleeding disorder caused by autoantibodies against factor VIII (FVIII). Hematological malignancies, especially lymphoid malignancies, are known to be underlying causes of AHA; however, thus far, there is no report of AHA associated with Epstein-Barr-virus-associated T/natural killer-cell lymphoproliferative disease (EBV-T/NK-LPD). Here, we present a case of AHA that developed during treatment for EBV-T/NK-LPD. HISTORY: A 69-year-old man visited our hospital because of general fatigue. Blood examination showed pancytopenia, and computed tomography revealed whole-body lymphadenopathy, but there were no findings indicating hematological malignancy from bone marrow aspiration and cervical lymph node biopsy. The level of EBV DNA in peripheral blood was extremely high, and he was diagnosed with EBV-T/NK-LPD. EBV-T/NK-LPD improved with prednisolone (PSL) administration. Seventeen months after starting treatment, the patient complained of back and right leg pain. At that time, he had been treated with low-dose PSL, and EBV-T/NK-LPD was well controlled. Imaging revealed hematoma of the right iliopsoas muscle. Prolonged activated partial thromboplastin time (APTT) was the only abnormal finding in a screening coagulation test. FVIII coagulant activity was below detection limit, and FVIII inhibitor level was increased. From these results, he was diagnosed with AHA.A higher dose of PSL was administered, and, after 1 month of treatment, FVIII activity gradually increased, and FVIII inhibitor level became undetectable. APTT also normalized, and complete remission was achieved and maintained for 13 months with low-dose PSL. During treatment, EBV-T/NK-LPD was well controlled. CONCLUSION: It is speculated that proliferating lymphocytes interfere with normal immune functions and that abnormal autoantibodies are produced from those lymphocytes in patients with LPD. Therefore, we speculate that EBV-infected and proliferating monoclonal NK cells might have modulated the immune system and produced autoantibodies against FVIII, thus causing AHA in this patient with EBV-T/NK-LPD.

Development of a Specific Monoclonal Antibody to Detect Male Cells Expressing the RPS4Y1 Protein.

Hemophilia is an X-linked recessive bleeding disorder. In pregnant women carrier of hemophilia, the fetal sex can be determined by non-invasive analysis of fetal DNA circulating in the maternal blood. However, in case of a male fetus, conventional invasive procedures are required for the diagnosis of hemophilia. Fetal cells, circulating in the maternal bloodstream, are an ideal target for a safe non-invasive prenatal diagnosis. Nevertheless, the small number of cells and the lack of specific fetal markers have been the most limiting factors for their isolation. We aimed to develop monoclonal antibodies (mAbs) against the ribosomal protein RPS4Y1 expressed in male cells. By Western blotting, immunoprecipitation and immunofluorescence analyses performed on cell lysates from male human hepatoma (HepG2) and female human embryonic kidney (HEK293) we developed and characterized a specific monoclonal antibody against the native form of the male RPS4Y1 protein that can distinguish male from female cells. The availability of the RPS4Y1-targeting monoclonal antibody should facilitate the development of novel methods for the reliable isolation of male fetal cells from the maternal blood and their future use for non-invasive prenatal diagnosis of X-linked inherited disease such as hemophilia.

B cell-activating factor modulates the factor VIII immune response in hemophilia A.

Inhibitors of factor VIII (FVIII) remain the most challenging complication of FVIII protein replacement therapy in hemophilia A (HA). Understanding the mechanisms that guide FVIII-specific B cell development could help identify therapeutic targets. The B cell-activating factor (BAFF) cytokine family is a key regulator of B cell differentiation in normal homeostasis and immune disorders. Thus, we used patient samples and mouse models to investigate the potential role of BAFF in modulating FVIII inhibitors. BAFF levels were elevated in pediatric and adult HA inhibitor patients and decreased to levels similar to those of noninhibitor controls after successful immune tolerance induction (ITI). Moreover, elevations in BAFF levels were seen in patients who failed to achieve FVIII tolerance with anti-CD20 antibody-mediated B cell depletion. In naive HA mice, prophylactic anti-BAFF antibody therapy prior to FVIII immunization prevented inhibitor formation and this tolerance was maintained despite FVIII exposure after immune reconstitution. In preimmunized HA mice, combination therapy with anti-CD20 and anti-BAFF antibodies dramatically reduced FVIII inhibitors via inhibition of FVIII-specific plasma cells. Our data suggest that BAFF may regulate the generation and maintenance of FVIII inhibitors and/or anti-FVIII B cells. Finally, anti-CD20/anti-BAFF combination therapy may be clinically useful for ITI.

Predicted coagulation potential using an in vitro simulated model of emicizumab prophylaxis and immune tolerance induction therapy in hemophilia A patients with inhibitor.

Emicizumab reduces bleeding in hemophilia A patients with inhibitor (HA-inh). A combination of immune tolerance induction therapy (ITI) and emicizumab prophylaxis may provide additional benefits, but coagulation potential during this treatment remains unknown. We assessed coagulation potentials in simulated ITI models in vitro using modified-clot waveform analysis. Factor (F)VIII-deficient plasma preincubated with anti-A2 and anti-C2 monoclonal antibodies was reacted with emicizumab (50 µg/mL) (emicizumab-HA-plasma), then spiking bypassing agents (BPAs): activated prothrombin complex concentrates (aPCC 1.3 IU/mL; 50 IU/kg), recombinant factor (rF)VIIa (2.2 µg/mL; 90 µg/kg), and FVIIa/FX (1.5 µg/mL; 60 µg/kg), and/or FVIII (100, 200 IU/dL). Coagulation potentials in emicizumab-HA-plasma (10 BU/mL) remained within the normal range when BPA and FVIII were both present. In emicizumab-HA-plasma (1 BU/mL) with BPA and FVIII (200 IU/dL), they were near or beyond the normal range, but those with a half concentration of rFVIIa based on the half-life in blood were within the normal range. In samples without inhibitor, coagulation potentials with combined BPA and FVIII were far beyond the normal range but with FVIII (100 IU/dL) and rFVIIa at half concentration they remained within the normal range. These results may provide information on the feasibility of concurrent ITI under emicizumab prophylaxis.

Development and desensitization therapy of high-response factor VIII inhibitors with severe allergic reaction in a moderate hemophilia A patient.

Neutralizing antibodies (inhibitors) against factor VIII/IX (FVIII/FIX) poses a serious and challenging complication in the hemophilia treatment. Allergic reaction is more common in hemophilia B and always companion with FIX inhibitors, but it is rare in hemophilia A (HA). So far only few cases demonstrated FVIII-specific allergic response in hemophilia A. Coexistence of allergic reactions with inhibitors was contraindicated for immune tolerance induction (ITI) regimen which is the only proven therapy to eliminate inhibitor. We report a rare case of a 11-year-old boy with moderate HA who developed high titer inhibitor and severe allergic reaction to both plasma derived and recombinant FVIII concentrates. Inhibitor was eliminated with the use of prednisone. Further desensitization protocol by administering rFVIII of increasing does from 0.01 IU/kg to 40 IU/kg with a pre-determined time schedule allowed patient tolerance to the normal dose and infusion time to FVIII.

Ex Vivo Prediction of Comprehensive Coagulation Potential Using Simulated Blood Concentrations of Emicizumab in Patients with Acquired Hemophilia A.

INTRODUCTION: Emicizumab prophylaxis improves coagulation function in congenital hemophilia A, regardless of inhibitor presence. We recently reported that emicizumab enhanced the coagulant potentials, ex vivo, in plasmas from patients with acquired hemophilia A (PwAHAs) at diagnosis. However, coagulant effects of emicizumab in PwAHAs during the clinical course remain unclear. AIM: To assess ex vivo coagulant effects of emicizumab in PwAHAs during the clinical course. METHODS/RESULTS: Blood samples were obtained from 14 PwAHAs on (median) days 0 and 6 during a severe-bleeding phase, and days 27 and 59 during a reduced-bleeding phase with elevated endogenous factor VIII (FVIII) and decreased inhibitor titers. If administered a single dose of 3 or 6 mg/kg, or two doses at 6 mg/kg followed by 3 mg/kg, estimated plasma emicizumab concentrations (10/5/2.5, 20/10/5, and 30/15/7.5 µg/mL on days 0-7/30/60, respectively) could be used to represent potential changes, based on the half-life (T 1/2: ∼30 days). Emicizumab concentrations that covered maximum plasma concentrations of each dosage were used for spiking on day 0. Ex vivo addition of estimated emicizumab to PwAHA's plasma containing endogenous FVIII and/or inhibitor, without and with recombinant (r)FVIIa administration during immunosuppressive therapy, increased the calculated Ad|min1| values, assessed by clot waveform analysis, and their coagulant potentials were below normal levels. Rotational thromboelastometry revealed that ex vivo emicizumab addition resulted in the further improvement of coagulant potentials in whole bloods when combined with rFVIIa administration. CONCLUSION: Based on ex vivo and in vitro data, emicizumab has the potential to be effective in clinical situations for PwAHAs.

Patients with haemophilia A with inhibitors in China: a national real-world analysis and follow-up.

The development of alloantibodies (inhibitors) against coagulation factor VIII (FVIII) is the most serious complication of FVIII replacement therapy in patients with haemophilia A (HA). We carried out a nationwide study focussing on patients with HA with inhibitors in China to evaluate the condition and management of this population. The study retrospectively analysed patient characteristics, clinical history, manifestation, treatment strategy as well as individual haemophilia care of 493 patients with inhibitors (466 with severe HA and 27 with non-severe HA) registered all over China. The median (interquartile range) age at diagnosis of FVIII inhibitors was 13 (5-28) years in patients with severe HA and 24 (10·5-39·5) years in patients with non-severe HA. Most patients (85%) had high-titre inhibitors. Prothrombin complex concentrate and recombinant activated coagulation factor VII were used respectively in 76·2% and 29·2% of patients for acute bleeding. Only 22·3% of patients underwent immune tolerance induction (ITI) treatment, of whom 64·9% achieved negative inhibitor titre. In patients who did not undergo ITI, the inhibitors turned negative in 17·7%, and patients with low peak inhibitor titre were more likely to acquire negative titre spontaneously (odds ratio 11·524, 95% confidence interval 5·222-25·432; P = 0·000). We recorded that 3·2% of the patients died from haemophilia-related life-threatening bleeding.