Electrocardiographic Findings Among Asymptomatic Adults With Haemophilia

Abstract Background: People with haemophilia (PwH) are living longer. Therefore, they can develop atherosclerotic cardiovascular disease (ASCVD). Electrocardiogram (ECG) alterations may be a sign of initial ASCVD before the occurrence of symptoms. Objective: To describe the prevalence of resting ECG alterations among PwH adults asymptomatic for ASCVD. Methods: PwH aged ≥ 30 years without previous ASCVD events were considered for the analysis. Resting ECG traces were analysed according to international reference values and the Brazilian Longitudinal Adult Health Study (ELSA-Brasil) results for asymptomatic Brazilian men. Based on the established normal values and using the QT index, we further described the altered ECGs as minor or major changes, according to the Minnesota Code. Differences between prevalences were evaluated by Pearson's χ2 test. Differences between medians were evaluated by the Mann-Whitney U test. A p-value < 0.05 was accepted as statistically significant. Results: A total of 64 PwH were included in the study. Median age was 44 years (interquartile range 35-52). Most patients had haemophilia A (81%) and 47% were severe. The prevalence of obesity, systemic arterial hypertension (SAH), diabetes mellitus (DM), and dyslipidaemia were 16%, 56%, 14%, and 72%, respectively. All the PwH had sinus rhythm, except for one, who had an implanted pacemaker due to idiopathic third-degree atrioventricular block. Altered ECGs were found in 25% and 30% of PwH, according to established criteria and ELSA-Brasil criteria, respectively. Major changes were found in eight (13%) PwH according to the Minnesota Code, including two ECGs with ischaemia-like wall inactivity. Conclusions: The prevalence of altered ECG varied from 25% to 30% among asymptomatic PwH.

Haemophilia A: A Review of Clinical Manifestations, Treatment, Mutations, and the Development of Inhibitors.

The purpose of this narrative review was to provide an overview that allows readers to improve their understanding of hemophilia A, which is considered a genetic disease with a high impact on the quality of life of people who suffer from it is considered one of the diseases with the highest cost for health systems (In Colombia it is part of the five diseases with the greatest economic impact). After this exhaustive review, we can see that the treatment of hemophilia is on the way to precision medicine, which involves genetic variables specific to each race and ethnicity, pharmacokinetics (PK), as well as environmental factors and lifestyle. Knowing the impact of each of these variables and their relationship with the efficacy of treatment (prophylaxis: regular infusion of the missing clotting factor VIII in order to prevent spontaneous bleeding) will allow for individualizing the medical behavior in a cost-effective way. For this is required to build more strong scientific evidence with statistical power that allows us to infer.

Socio-emotional repercussions of severe haemophilia A in the daily lives of children.

INTRODUCTION AND AIM: Haemophilia A, in its most severe form, can have serious repercussions, including issues that are physical, emotional, affective, and social, particularly in childhood. This qualitative study aims to understand the socio-emotional repercussions of severe haemophilia A in children, based on their own testimonies and subjective expressions of their daily lives, in the contexts of the family, school and health service. METHODS: Individual qualitative interviews were carried out using a playful approach through puppets with 15 children, aged 6-12 years old, in a service for the treatment of haemophilia, located in the northeast of Brazil. Data were analysed using inductive thematic analysis. RESULTS: Four themes were elaborated: (a) Reflecting how I am and how I relate to others; (b) Enjoying family moments; (c) Experiencing the school context: learning, affectivity and play; and (d) Dealing with haemophilia: acceptance and overcoming strategies. CONCLUSION: The experiences shared by children with severe haemophilia A and their daily needs should be the basis for guiding child-centred care. Encouraging self-care, including self-administration of the deficient factor, requires a partnership between health professionals, family members, school and child in the construction of therapeutic plans that consider the child's active participation.

Molecular study of a large cohort of 109 haemophilia patients from Cuba using a gene panel with next generation sequencing-based technology.

INTRODUCTION: In several countries, molecular diagnosis of haemophilia A (HA) and B (HB) is hampered by a lack of resources for DNA analysis. The advent of next-generation sequencing (NGS) has enabled gene analysis at a reasonable cost. AIM: Describe a collaboration between Cuban and Spanish researchers to identify candidate variants and investigate the molecular epidemiology of 106 Cuban haemophilia patients using NGS. PATIENTS/METHODS: The molecular analysis protocol included well-established LR-PCR procedures to detect F8 inversions, NGS with a 30-gene panel to sequence F8 and F9, and multiplex ligation-dependent probe amplification to identify large structural variants. RESULTS: One-hundred and thirty-one candidate variants were identified along F8, F9, and VWF; 72 were unique and 28 (39%) had not been previously recorded. Putative variants were identified in 105/106 patients. Molecular characterization enabled confirmation and reclassification of: 90 HA (85%), 15 HB (14%), and one type 2N VWD (1%). Null variants leading to non-production of FVIII or FIX were common in severe HA (64%), moderate HA (74%), and severe HB (60%), whereas missense variants were frequent in mild HA (57%) and moderate or mild HB (83%). Additional variants in VWF were identified in 16 patients. CONCLUSION: This is the first description of the molecular epidemiology of HA and HB in Cuba. Variants identified in index cases will be of value for local implementation of familial studies and prenatal diagnosis using the molecular approaches available in Cuba. The results of this protocolled genetic study improved the accuracy of the clinical diagnosis and will facilitate management of these patients.

Personalised Prophylaxis in a Child with Haemophilia A and Type 1 Diabetes.

Poor management of either type 1 diabetes or haemophilia A can lead to complications such as organ dysfunction and haemarthropathy. Here, we describe the case of an 8-year-old boy diagnosed with severe haemophilia A shortly after birth. At 2 years old, he was also diagnosed with type 1 diabetes. After six years, the haemophilia treatment was changed from a plasma-derived factor VIII (FVIII) concentrate (octanate®, Octapharma, Lachen, Switzerland) to Nuwiq® (simocotocog alfa, Octapharma, Lachen, Switzerland), a recombinant FVIII (rFVIII) product from a human cell line, which allowed for a personalised treatment schedule that supported good adherence. The dosing regimen could be reduced to two weekly rFVIII infusions. The patient has experienced no spontaneous bleeds since switching to rFVIII and shows no signs of joint damage after over seven years of FVIII prophylaxis. rFVIII was well tolerated, with no treatment-related adverse events observed. This case illustrates the importance of treatment personalisation for young patients and their families managing concomitant diseases.

Functional and joint evaluation in a prospective cohort of patients with severe haemophilia.

INTRODUCTION: Arthropathy is the main morbidity of haemophilia. Periodic joint health assessment enables a better understanding of the limitations of these patients. OBJECTIVE: To evaluate the functional and joint performance in patients with haemophilia at one-year follow-up, as well as its association with prophylactic treatment and attendance at scheduled consultations at a haemophilia treatment centre. METHODS: This prospective cohort study included patients with severe haemophilia aged 7 years or more and treated at Fundação Hemocentro de Brasília, Brazil, from January 2014 to December 2018. The Hemophilia Joint Health Score and Functional Independence Score in Hemophilia were assessed at the first consultation and after a one-year follow-up. RESULTS: The study included 69 patients. The mean age at study recruitment was 22.5 ± 4.5 years, 62.3% of patients aged 18 years or older, and 29 patients were receiving primary prophylaxis (38.0%). There was a positive correlation between HJHS and age and a negative correlation between FISH and age. The worsening HJHS was associated with non-primary prophylaxis and non-attendance at scheduled multidisciplinary consultations. The worsening FISH was associated with non-primary prophylaxis. The correlation between FISH and treatment adherence was significant for the delta. CONCLUSION: The older the patient with haemophilia, the higher the probability of a worsening of the HJHS. In the presence of more arthropathies, the older the patient, the worse the FISH. Patients receiving primary prophylaxis show better results in the HJHS and FISH when compared to patients receiving secondary prophylaxis and/or on-demand treatment.

Consenso de hemofilia en México / Mexican Consensus in Haemophilia

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Abstract Hemophilia is a hemorrhagic disorder with a sex-linked inherited pattern, characterized by an inability to amplify coagulation due to a deficiency in coagulation factor VIII (hemophilia A or classic) or factor IX (hemophilia B). Sequencing of the genes involved in hemophilia has provided a description and record of the main mutations, as well as a correlation with the various degrees of severity. Hemorrhagic manifestations are related to levels of circulating factor, mainly affecting the musculoskeletal system and specifically the large joints (knees, ankles and elbows). This document is a review and consensus of the main genetic aspects of hemophilia, from the inheritance pattern to the concept of women carriers, physiopathology and classification of the disorder, the basic and confirmation studies when hemophilia is suspected, the various treatment regimens based on infusion of the deficient coagulation factor as well as innovative factor-free therapies and recommendations for the management of complications associated with treatment (development of inhibitors and/or transfusion transmitted infections) or secondary to articular hemorrhagic events (hemophilic arthropathy). Finally, relevant reviews of clinical and treatment aspects of hemorrhagic pathology charachterized by acquired deficiency of FVIII secondary to neutralized antibodies named acquired hemophilia.

Primera experiencia de tratamiento con emicizumab en un paciente con hemofilia A severa e inhibidor de alto título en Uruguay / First report of emicizumab use in a patient with severe haemophilia A and high-titer inhibitor in Uruguay / Primeira experiência de tratamento com emicizumab em paciente com hemofilia A grave e inibidor de alto título no Uruguai

La hemofilia A es una coagulopatía congénita causada por la deficiencia o el mal funcionamiento del factor VIII de la coagulación. Una de las complicaciones más graves del tratamiento de la hemofilia A es el desarrollo de inhibidores que hacen que la terapia de reemplazo con FVIII sea ineficaz, dificultando la prevención y el control de los sangrados. El emicizumab es un anticuerpo monoclonal humanizado biespecífico dirigido contra los factores FIXa y FX, que imita la función de cofactor del FVIII. El tratamiento profiláctico con emicizumab es seguro y eficaz para prevenir hemorragias en los pacientes con hemofilia A con y sin inhibidores. Se presenta el caso del primer paciente tratado con emicizumab en Uruguay.

Haemophilia A is a congenital coagulopathy caused by a deficiency or malfunction of coagulation factor VIII. One of the most serious complications of haemophilia A treatment is the development of inhibitors that render FVIII replacement therapy ineffective, making it difficult to prevent and control bleeding. Emicizumab is a humanized bispecific monoclonal antibody directed against factors FIXa and FX, which mimics the cofactor function of FVIII. Emicizumab has been shown to be safe and effective as prophylaxis to prevent bleeding in haemophilia A patients with or without inhibitors to FVIII. We report the first patient treated with emicizumab in Uruguay.

A hemofilia A é uma coagulopatia congênita que se caracteriza pela ausência ou mau funcionamento do factor VIII da coagulação. Uma das complicações mais sérias do tratamento da hemofilia A é o desenvolvimento de inibidores que tornam a terapia de reposição do FVIII ineficaz, dificultando a prevenção e o controle do sangramento. O emicizumab é um anticorpo monoclonal biespecífico humanizado dirigido contra os fatores FIXa e FX, que imita a função de cofator do FVIII. O tratamento profilático com emicizumab é seguro e eficaz na prevenção de sangramento em pacientes com hemofilia A com e sem inibidores. É apresentado o caso do primeiro paciente tratado com emicizumabe no Uruguai.

Validación del ensayo FVIII: C cromogénico en una plataforma automatizada

El objetivo de este trabajo fue realizar la validación analítica del método cromogénico (FVIII:Ccro) en la plataforma ACL TOP y correlacionarlo con el método coagulable en una etapa (FVIII:Ccoag). El estudio de validación (EP5-A2, EP6-A2 y comparación de métodos por EP-9) se realizó para la curva de rango normal-bajo (CRNB): aproximadamente entre 10-150 UI/dL de FVIII y de rango muy bajo (CRMB): aproximadamente entre 0-10 UI/dL. Los resultados de repetitividad (CVr) y precisión intermedia (CVi) fueron menores del 6% y comparables a los informados por el fabricante para otras plataformas. El rango de medición analítica fue de 11-129 UI/dL con CRNB, y se extrapoló a 0,3 UI/dL al utilizar la CRMB. Para la CRNB FVIII:Ccro mostró buena correlación con FVIII:Ccoag: r: 0,98, pendiente: 0,982 (0,961-1,003), ordenada al origen: -0,3 (-1,1-0,5), sesgo: -2,0%. Para CRMB se obtuvo un r de 0,96, pendiente: 0,921 (0,855-0,988), ordenada al origen: -0,07 (-0,35-0,20), sesgo: -10,2%. Sólo 4 pacientes presentaron niveles discrepantes entre ambos métodos. La determinación de FVIII:C por el método cromogénico automatizado en la familia ACL TOP fue comparable con FVIII coagulable en una etapa en el rango analítico evaluado. El FVIII:Ccro automatizado puede utilizarse para el diagnóstico y seguimiento del tratamiento de los pacientes hemofílicos.

The objective of this work was to perform the analytical validation of the chromogenic method (FVIII:Ccro) on the ACL TOP platform correlating with one stage assay (FVIII:Ccoag). The validation study (EP5-A2, EP6-A2 and comparison of methods by EP-9) was performed for the low-normal range curve (CRNB): approximately between 10-150 IU/dL of FVIII and very low range (CRMB): approximately between 0-10 IU/dL. The results of CVr (repeatability) and CVi (intermediate precision) were lower than 6% and comparable to those reported by the manufacturer for other platforms. The analytical measurement range was 11-129 IU/dL, extrapolated to 0.3 IU/dL using the CRMB. For CRNB FVIII:Ccro showed good correlation with FVIII:Ccoag: r: 0.98, slope: 0.982 (0.961-1.003), intercept: -0.3 (-1.1-0.5), bias: -2.0%. For CRMB: r: 0.96 was obtained, pending: 0.921 (0.855-0.988), intercept: -0.07 (-0.35-0.20), bias: -10.2%. Only 4 patients presented discrepant levels between both methods. The automated chromogenic FVIII assay in the ACL TOP family is comparable with one stage coagulable FVIII in the analytical range studied. The FVIII:Ccro automated can be used for the diagnosis and monitoring of the treatment of hemophilic patients.

O objetivo deste trabalho foi a validação analítica do método cromogênico (FVIII:Ccro) na plataforma ACL TOP correlacionando-se com o método coagulável numa etapa (FVIII:Ccoag). O estudo de validação (EP5-A2, EP6-A2 e comparação de métodos por EP-9) foi realizado para a curva de faixa normal-baixa (CRNB) aproximadamente entre 10 e 150 Ul/dL de FVIII e de faixa muito baixa (CRMB): aproximadamente entre 0 e 10 UI/dL. Os resultados de Repetitividade (CVr) e precisão intermediária (CVi) foram inferiores a 6% e comparáveis aos descritos pelo fabricante para outras plataformas. A faixa de medição analítica foi de 11-129 UI/dL com CRNB extrapolando-se para 0,3 UI/dL utilizando a CRMB. Para a CRNB FVIII:Ccro houve boa correlação com o FVIII: Ccoag: r: 0,98, inclinação: 0,982 (0,961-1,003), ordenada na origem: -0,3 (-1,1-0,5), Viés: -2,0%. Para CRMB: foi obtido um r: 0,96, pendente: 0,921 (0,855-0,988), ordenado na origem: -0,07 (-0,35-0,20), Viés: -10,2%. Apenas quatro pacientes apresentaram níveis discrepantes entre os dois métodos. A determinação de FVIII:C pelo método cromogênico automatizado na família ACL TOP foi comparável ao FVIII coagulável em um estágio na faixa analítica avaliada. FVIII: O FVIII:Ccro automatizado pode ser utilizado para o diagnóstico e seguimento do tratamento dos pacientes hemofílicos.

Molecular genetic diagnosis by next-generation sequencing in a cohort of Mexican patients with haemophilia and report of novel variants.

INTRODUCTION: Molecular analysis in haemophilia is currently used in the diagnosis, treatment and prognosis of this disease. Hispanic populations in Latin America have been of interest to researchers due to the reportedly high prevalence of inhibitors in these patients. AIM: To perform next-generation sequencing (NGS) in a cohort of Mexican patients with HA and HB and correlate with clinical phenotypes. METHODS: Patients with Haemophilia A (HA) or haemophilia B (HB), were evaluated using NGS with an Ion AmpliSeq Custom Panel. Odds ratios (ORs) for associations between F8 variants and inhibitors were obtained. RESULTS: A total of 85 patients (60 with HA and 25 with HB) were included. Pathogenic variants in F8 were found in 93.3% of HA patients and in F9 in 96% of HB patients. Twelve novel potentially pathogenic variants were found. Inhibitors were observed in 20% of patients with severe HA. Four patients clinically diagnosed with HA were negative for F8 variants. CONCLUSION: Overall detection rate of pathogenic variants in F8 and F9 genes was 94.6%. We identified 12 non previously reported variants and pathogenic variants in other coagulation related genes. Molecular diagnosis of HA and HB permits better options for management, assessment and genetic counseling.

Manejo quirúrgico de pseudotumor hemofílico retroperitoneal: Reporte de caso / Surgical management of a retroperitoneal hemophilic pseudotumor: Report of a case

El pseudotumor hemofílico es una complicación muy rara vista en pacientes con hemofilia severa, y que cuyo manejo aún no se encuentra estandarizado. Se presenta el caso de un paciente varón de 33 años de edad admitido en el servicio de cirugía del Hospital Nacional Dos de Mayo por presentar una tumoración localizada en fosa iliaca izquierda, dolorosa a la palpación, asociada a alteración de la marcha. Realizada la exéresis, se identificó un pseudotumor hemofílico gigante ubicado en retroperitoneo. Este caso expone el primer reporte de manejo de pseudotumor hemofílico en nuestro país, con resultados satisfactorios a los 12 meses del posoperatorio.

The hemophilic pseudotumor is a very rare complication occurring in patients with severe hemophilia, and its management has not been standardized yet. We present the case of a 33-year old male patient who was admitted to the surgery ward in Dos de Mayo Hospital in Lima, Peru, who presented with a tumoral mass located in the left iliac fossa, which was painful when palpated, and it was associated with gait alterations. When the mass was excised, a giant hemophilic pseudotumor located in the retroperitoneum was identified. This is the first case describing the management of a hemophilic pseudotumor in our country, with satisfactory results at 12 months after surgery.

Immune status of patients with haemophilia A before exposure to factor VIII: first results from the HEMFIL study.

Previous cross-sectional studies showed that some patients with haemophilia A (HA) without inhibitor presented a pro-inflammatory profile during factor VIII (FVIII) replacement therapy. Furthermore, an anti-inflammatory/regulatory state was described in HA patients after inhibitor development. However, no study investigated the levels of these biomarkers before exposure to exogenous FVIII. This study investigated the immunological profile of previously untreated patients (PUPs) with HA in comparison with non-haemophiliac boys. A panel of chemokines and cytokines was evaluated in the plasma of 40 PUPs with HA and 47 healthy controls. The presence of microparticles was assessed in the plasma of 32 PUPs with HA and 47 healthy controls. PUPs with HA presented higher levels of CXCL8 (IL8), IL6, IL4, IL10, IL2, IL17A (IL17), and lower levels of CXCL10 (IP-10) and CCL2 (MCP-1) than the age-matched healthy controls (P < 0·05). We also observed higher levels of microparticles derived from endothelium, erythrocytes, platelets, leucocytes, neutrophils, and T lymphocytes in patients in comparison with controls (P < 0·05). Compared with controls, PUPs with HA presented a distinct immunological profile, characterized by a prominent pro-inflammatory status that appears to be regulated by IL4 and IL10.

Comparación de la conducta de enfermedad en pacientes colombianos con hemofilia A, en una muestra con dolor crónico y sin dolor crónico / Comparison of disease behavior in Colombian patients with hemophilia A, in a sample compared with chronic pain and without chronic pain

Objetivo. Identificar las variables de conducta de enfermedad en una muestra de pacientes colombianos con Hemofilia A y comparar los resultados de grupos (con dolor crónico y sin dolor crónico) para un análisis de relaciones entre variables. Método. El enfoque fue cuantitativo y su diseño transversal; la metodología empleada fue ex post facto de tipo retrospectiva. Para el análisis estadístico se hizo una comparación de grupos (sin dolor crónico y con dolor crónico). El factor de conducta de enfermedad se evaluó a través del Cuestionario de Conducta de Enfermedad de Ballester y Botella del año 1993. Los resultados obtenidos mediante la prueba T, la correlación de Spearman, la prueba U de Mann-Whitney y la Curva de ROC. Resultados. Se evaluaron veintisiete sujetos con diagnóstico de Hemofilia A con un rango de edad entre 19 y 55 años. Diecisiete sujetos conformaron el grupo con dolor crónico y diez sujetos el grupo sin dolor crónico. En conclusión se hallaron asociaciones significativas entre el dolor crónico y las variables relacionadas a inestabilidad emocional, así como correlaciones positivas con resultados de investigaciones previas sobre la caracterización de la población con hemofilia.

Objective. To Identify the variables of illness behavior in a sample of Colombian patients with hemophilia A and to compare the results of groups (with chronic pain and without chronic pain chronic) for an analysis of relationships between variables. Methods. The approach was quantitative and its cross-sectional design. The methodology used was ex post facto retrospective type. A comparison groups (without chronic pain and chronic pain) was done for the statistical analysis. The disease behavior factor was evaluated through the Ballester and Botella Disease Questionnaire Conduct of 1993. The results obtained by the T-test, the Spearman correlation, the Mann-Whitney U test and the ROC curve. Results. Twenty-seven subjects diagnosed with haemophilia A diagnosed with a age range between 19 and 55 years were evaluated. Seventeen subjects formed the group with chronic pain and ten subjects the group without chronic pain. In conclusion, we found significant associations between chronic pain and variables related to emotional instability, as well as positive correlations with results of previous research on the characterization of the population with hemophilia.

Updates from guardian™: a comprehensive registration programme.

Turoctocog alfa is an approved B-domain truncated recombinant factor VIII concentrate for adults and children with haemophilia A. Clinical data for turoctocog alfa have been reported from the guardian(™) 1, guardian(™) 2 and guardian(™) 3 phase III trials. guardian(™) 1 and guardian(™) 3 phase III trials enrolled 150 adolescents/adults (≥ 12 yr), and 63 children (<12 yr), respectively, with previously treated severe haemophilia A and no history of inhibitors; 188 of these patients continued into the ongoing guardian(™) 2 extension trial. In the three trials, patients have received prophylaxis with turoctocog alfa three times weekly or every second day, with breakthrough bleeds resolved to an expected postinjection level of ≥ 0.50 IU/mL. No safety concerns have arisen, and no patients have developed confirmed inhibitors (primary endpoint). Indeed, no confirmed inhibitors have been detected in >200 patients treated for a cumulative total of >54,000 exposure days in the phase III trials. Pooled efficacy data show a favourable long-term effect of turoctocog alfa on annualised bleeding rate and a success rate for haemostatic response of 86%; 90% of bleeds were resolved with 1-2 doses. This article reviews the clinical development of turoctocog alfa with reference to the guardian(™) clinical programme, describing results obtained to date and ongoing trials.

Allergic reaction in a cohort of haemophilia A patients using plasma-derived factor VIII (FVIII) concentrate is rare and not necessarily triggered by FVIII.

In contrast to haemophilia B, allergic manifestations are rare complications in haemophilia A (HA) patients treated with factor VIII (FVIII) concentrates. Nevertheless, it can be serious and hamper replacement therapy in these cases. The aims of this study were to evaluate the frequency of allergic reaction in a cohort of HA patients treated only with plasma-derived FVIII (pdFVIII) concentrates, and assess the possible immune mechanisms involved. History of allergic reaction was retrospectively assessed. Patients with allergic manifestations were followed, and had plasma samples collected in different timepoints in relation to the allergic episode. These samples were analysed for the presence of inhibitor and anti-FVIII immunoglobulins subclasses. Three of 322 HA patients (0.9%) developed allergic reaction after exposure to pdFVIII products during the last 15 years in our centre. The first patient, with severe HA, without inhibitor, had anti-pdFVIII IgE and IgG4, but no anti-recombinant FVIII (rFVIII) IgE. The second patient, with severe HA, and high-responding inhibitor, presented allergic manifestation with both, pdFVIII concentrate and activated prothrombin complex concentrate. Although anti-pdFVIII and anti-rFVIII IgG4 were detected, no anti-FVIII IgE was present. The third patient, with moderate HA without inhibitor, atopic, had no anti-FVIII immunoglobulin detected, and allergic symptoms disappeared after switching to rFVIII concentrate. This study corroborates the low incidence of allergic reactions in HA patients. In the three cases presented, the anti-FVIII immunoglobulin profile demonstrated that the allergic manifestation was triggered by other proteins contained in pdFVIII products, and not directed to FVIII.

New associations: INFG and TGFB1 genes and the inhibitor development in severe haemophilia A.

INTRODUCTION: The development of factor VIII (FVIII) inhibitor is the main complication of replacement therapy in patients with haemophilia A (HA). A ratio of 5-7% of individuals HA develops antibodies (inhibitors) against the FVIII infused during the treatment, thereby reducing their pro-coagulant activity. The immunomodulatory cytokine genes have been related to the risk of development of alloantibodies in several studies, mainly in HA with severe form. AIM: We investigated the polymorphisms in regulatory regions of cytokine genes (IL1A, IL1B, IL1R, IL1RA, IL4RA, IL12, INFG, TGFB1, TNF, IL2, IL4, IL6, IL10) that could influence the risk of developing inhibitors in patients with severe HA. METHODS: The genotyping of cytokine genes of 117 patients with HA was performed by polymerase chain reaction with sequence-specific primers (PCR-SSP) using the protocol recommended by the manufacturer (Invitrogen kit Cytokines(®) , Canoga Park, USA) RESULTS: From the cohort of 117 patients with severe HA, 35 developed inhibitors. There was a higher frequency of +874 T allele in INFG and of +869 TT and TG/TG in TGFB1 genes on patients with inhibitors. CONCLUSION: This suggests that polymorphisms in INFG and in TGFB1 genes are related to risk of developing inhibitor, and could contribute to a genetic profile of the individual HA for the risk of inhibitors development to FVIII.

A quantitative and humane tail bleeding assay for efficacy evaluation of antihaemophilic factors in haemophilia A mice.

The tail bleeding model using haemophilic mice has been used as one of the standard assays for efficacy evaluation of novel antihaemophilic therapies at the preclinical level. A number of different configurations and endpoints have been proposed in the literature for this model, hindering interlaboratory comparisons. A particular configuration, known as the tail bleeding survival assay (TBS), adopted by several groups, involves measuring the ability of conscious haemophilic mice to survive exsanguination following tail transection. Major limitations to this configuration include ethical constraints and impaired quantitative determinations. The aim of this study was to standardize and validate a quantitative haemostatic assay for evaluation of antihaemophilic therapies employing an alternative to TBS, which involves a more humane endpoint associated with stable clot formation. Haemophilic mice were treated with vehicle or different doses of two antihaemophilic reference products licensed in Brazil. The haemostatic response was evaluated by our quantitative tail bleeding haemostatic assay (qTBA) over a period of 120 min and then quantified by dose-response modelling. We demonstrate that our qTBA method allows a direct relationship between the number of animals which achieved full haemostatic response and the dosage of both antihaemophilic factors evaluated over 120 min. In addition, the method sensitivity is suitable to demonstrate the conversion from a severe to a moderate haemophilia phenotype. Our proposed qTBA is easy to implement and constitutes an alternative and more ethical endpoint, which could be effectively used as a surrogate to the commonly employed survival endpoint, allowing quantitative haemostatic response evaluation associated with stable clot formation.

Genetic testing in bleeding disorders.

The aim of molecular genetic analysis in families with haemophilia is to identify the causative mutation in an affected male as this provides valuable information for the patient and his relatives. For the patient, mutation identification may highlight inhibitor development risk or discrepancy between different factor VIII assays. For female relatives, knowledge of the familial mutation can facilitate carrier status determination and prenatal diagnosis. Recent advances in understanding mutations responsible for haemophilia and methods for their detection are presented. For reporting of such mutations, participation in external quality assessment ensures that essential patient and mutation details are routinely included and that pertinent information is incorporated in the interpretation.

Randomized comparison of prophylaxis and on-demand regimens with FEIBA NF in the treatment of haemophilia A and B with inhibitors.

Factor replacement therapy for the treatment of moderate to severe haemophilia A and B can be complicated by the production of inhibitory alloantibodies to factor VIII (FVIII) or factor IX. Treatment with the nanofiltered anti-inhibitor coagulant complex, Factor Eight Inhibitor Bypassing Activity (FEIBA NF), is a key therapeutic option for controlling acute haemorrhages in patients with high-titre inhibitors or low-titre inhibitors refractory to replacement therapy. Given the high risk for morbidity and mortality in haemophilia patients with inhibitors to FVIII or FIX, we conducted this Phase 3 prospective study to evaluate whether prophylaxis with FEIBA NF is a safe and effective treatment option. Over a 1-year period, 17 subjects were treated prophylactically (85 ± 15 U kg(-1) every other day) while 19 subjects were treated on demand. The median (IQR) annualized bleeding rate (ABR) during prophylaxis was 7.9 (8.1), compared to 28.7 (32.3) during on-demand treatment, which amounts to a 72.5% reduction and a statistically significant difference in ABRs between arms (P = 0.0003). Three (17.6%) subjects (ITT) on prophylaxis experienced no bleeding episodes, whereas none treated on demand were bleeding episode-free. Total utilization of FEIBA NF for the treatment of bleeding episodes was significantly higher during on-demand therapy than prophylaxis (P = 0.0067). There were no differences in the rates of related adverse events between arms. This study demonstrates that FEIBA prophylaxis significantly reduces all types of bleeding compared with on-demand treatment, and the safety of prophylaxis is comparable to that of on-demand treatment.

Detection of new mutations and molecular pathology of mild and moderate haemophilia A patients from southern Brazil.

A total of 76 unrelated male patients with mild (n = 55) or moderate (n = 21) haemophilia A living in the southern Brazilian state of Rio Grande do Sul were studied by direct sequencing of all F8 26 exons, the 5' UTR and 3' UTR, intron-exon junctions and the promoter region. When no mutation was found, a multiplex ligation-dependent probe amplification analysis was performed. We identified the disease-causing mutations in 69 patients, who showed 33 different mutations: 27 missense, one small deletion, two small duplications and three splice site mutations. Seven missense and two splice site mutations were not previously reported in HAMSTeRS and were not identified in any current literature search. Nine recurrent mutations were found, one of them never described before (p.Tyr1786Phe). Haplotype analysis indicated that this mutation had originated in the Brazilian population as a single event in a common ancestor. The possible influence of these mutations in the determination of the disease was carefully considered, including bioinformatic tools. These data add to the general knowledge of the disease and can also be useful for HA diagnosis and detection of carriers in the southern Brazilian population.