Inhibitor incidence after intensive FVIII replacement for surgery in mild and moderate haemophilia A: a prospective national study in the Netherlands

Inhibitor development is currently the most severe complication in mild/moderate haemophilia A patients, causing increased bleeding tendency, hospitalization and mortality. It has been suggested that receiving high doses of factor VIII (FVIII) concentrates for surgical procedures is an important risk factor for inhibitor development in these patients. The current multicentre study aimed to determine prospectively the incidence of inhibitor development after intensive FVIII replacement therapy for surgical procedures in patients with mild/moderate haemophilia A. All consecutive patients with mild/moderate haemophilia A were included when they required at least 10 000 iu of FVIII concentrates (or 250 iu/kg) for 5 or more days for a surgical procedure. Potential clinical risk factors for inhibitor development and results of inhibitor tests were collected. Forty-six patients with a median age of 54 years (interquartile range, 40-59 years) were included in the study. F8 genotyping revealed 20 different missense mutations. Patients received either recombinant (65%) or plasma-derived FVIII concentrates (35%) by intermittent bolus injections (41%) or continuous infusion (57%). Two patients developed a low titre inhibitor post-operatively. The incidence of inhibitor development following intensive treatment for surgery in this unselected prospective cohort of mild/moderate haemophilia A patients was 4% (95% confidence interval, 0·5-14·8).     

Risk of inhibitor development in mild haemophilia A increases with age

Summary. Mild haemophilia A is a rare disease with a relatively mild phenotype. Treatment with factor VIII (FVIII) is indicated after trauma or for surgery only. FVIII infusion may result in the development of inhibiting antibodies against FVIII. This study describes the relation between age and other risk factors for inhibitor development in mild haemophilia. A retrospective cohort study was conducted among all patients with mild haemophilia (FVIII 0.05–0.40 IU mL^?1) registered at the van Creveldkliniek, University Medical Centre Utrecht, The Netherlands. Data on peak treatment with FVIII, gene mutation and history of inhibitor development were obtained from patient files from the period between 1st January 1970 and 31st December 2009. A total of 231 out of 297 (78%) patients had at least one exposure to FVIII, of whom 14 (6.1%) developed an inhibitor to FVIII at a median age of 66 years after a median of 50 exposure days (ED). Age at first exposure, age at peak treatment, number of peak treatments and Arg593Cys mutation were significantly associated with the development of an inhibitor, while continuous infusion with FVIII was not. Although the incidence of inhibitors in mild haemophilia is low, it increases with age and peak treatments. With increasing age patients with mild haemophilia will suffer from co‐morbidity more frequently, requiring surgical interventions and exposing them to an increased risk of inhibitor development. Especially patients with a change of arginine in cysteine at 593 are at risk for inhibitor development.     

Hydrolysis of factor VIII mediated by catalytic antibodies occurs in haemophilia A patients with or without factor VIII inhibitors

The major complication of the substitutive treatment of haemophilia A (HA) is the development of antifactor VIII (FVIII) antibodies. Most of these antibodies neutralize FVIII procoagulant activity, and are identified as FVIII inhibitor. A subgroup of these antibodies, ‘catalytic antibodies’, catalyses the FVIII hydrolysis. We investigated the frequency and the activity of catalytic antibodies, according to the phenotype of HA and the presence or absence of FVIII inhibitor. IgG from 16 patients with inhibitor and 17 patients without inhibitor were purified. Rates of FVIII hydrolysis and inhibitor titres were evaluated. Anti‐FVIII catalytic antibodies were detected in 63.6% of patients with HA, irrespective of the HA phenotype and the presence of FVIII inhibitor. The frequency was significantly higher for severe HA patients (73.3%) and patients with inhibitor (87.5%), but their FVIII‐proteolytic activity was not significantly different from patients with mild or moderate HA and patients without inhibitor. The evolution of both catalytic and inhibitory activities was studied for 11 patients with FVIII inhibitor. We observed two profiles. In the profile 1, 18.2% of patients, the catalytic activity and the inhibitor titre coevolved. In contrast, a dissociated evolution of these two parameters was observed in 72.8% patients in profile 2. These data confirm the importance of anti‐FVIII catalytic activity in patients with severe, moderate and mild HA. Interestingly, most of the patients presented a dissociated profile, suggesting that anti‐FVIII antibodies might not systematically act as FVIII inhibitors.     

Protected by nature? Effects of strenuous physical exercise on FVIII activity in moderate and mild haemophilia A patients: a pilot study

Increase of factor VIII activity (FVIII) after physical exercise has been reported in healthy subjects and small‐scale studies in patients with coagulopathies. The aim was to study whether moderate and mild haemophilia A patients are able to increase their endogenous FVIII activity levels by physical activity. We studied changes in FVIII activity levels after high‐intensity exercise in 15 haemophilia A patients, 20–39 years, eight with moderate, seven with mild haemophilia. Patients cycled until volitional exhaustion, blood samples were drawn before and 10 min after the exercise test. FVIII activity increased 2.5 times (range 1.8–7.0 times), for both severities. Absolute increases were markedly different: median 7 IU dL^?1 (range 3–9 IU dL^?1) in patients with moderate, compared to 15 IU dL^?1 (range 6–62 IU dL^?1) in mild haemophilia patients. VWF and VWFpp increased independently of severity; median 50% (range 8–123%) and median 165% (range 48–350%), respectively, reflecting acute release of VWF. These observations may be used to promote high‐intensity activities before participating in sports for moderate and mild haemophilia A patients, to reduce bleeding risk. Further studies are warranted to fully appreciate the clinical significance of exercise on different levels of intensity in patients with mild and moderate haemophilia A.     

Clinical severity of haemophilia A: does the classification of the 1950s still stand?

Summary. The classification of haemophilia originates from 1950s and has been adopted unchallengedly by the ISTH in 2001. The aim of this study was: does the current classification compare onset of bleeding and age at first treatment, as well as annual joint bleeding frequency according to baseline FVIII activity? Data on age and reason of diagnosis, onset of treatment, onset of bleeding and bleeding frequency from 411 patients with haemophilia A born after 1970 were collected. Data were analysed according to base‐line FVIII activity levels. Age at diagnosis, onset of bleeding and start of treatment according to FVIII activity were compared with the current classification. Overall, the distinction between severe and non‐severe haemophilia was clear. The distinction between mild and moderate haemophilia was more difficult, mostly due to the wide variability in the group of patients with moderate haemophilia. Patients with severe haemophilia experienced their milestones like diagnosis, first treatment and joint bleed earliest, mostly as infants aged 0–3 years, whereas patients with moderate haemophilia reached these milestones around toddler age, 2–7 years, and patients with mild haemophilia reached them when they were in elementary school, around the ages of 5–14 years. This study confirms the clinical distinction between severe and non‐severe haemophilia A. However, the group of moderate haemophilia patients showed a wide variability, warranting close follow‐up and individualized treatment.     

A longitudinal evaluation of anti‐FVIII antibodies demonstrated IgG4 subclass is mainly correlated with high‐titre inhibitor in haemophilia A patients

The development of inhibitory antibodies against factor VIII (FVIII) (inhibitor) is the major complication in haemophilia A patients. The FVIII‐binding antibodies development comprises a polyclonal immunoglobulin (Ig) G response. Recent studies showed strong correlation between the presence of neutralizing anti‐FVIII antibodies (inhibitors) and IgG4 subclass. The aim of this study was to evaluate anti‐FVIII IgG subclasses in haemophilia A patients with inhibitor both in a cross‐sectional and in a longitudinal analysis. Inhibitors were determined by Nijmegen–Bethesda assay. Anti‐FVIII IgG subclasses were performed by ELISA, and samples from 20 healthy individuals were used to validate the test. We studied 25 haemophilia A patients with inhibitor, previously treated exclusively with plasma‐derived FVIII concentrates or bypassing agents. The IgG subclasses distributions were evaluated in two groups of patients classified according to inhibitor response. IgG1 and IgG4 antibodies were most prominent in haemophilia A patients with inhibitors when compared with IgG2 and IgG3. This study reports for the first time the behaviour of FVIII‐binding IgG1 and IgG4 subclasses in a longitudinal analysis, in a clinical setting, of high‐response inhibitor haemophilia A patients, showing the correlation of IgG4 and the inhibitor titres. In spite of being considered a non‐pathologic antibody subclass with anti‐inflammatory properties in other situations, IgG4 is correlated with the presence of high‐titre inhibitor in the haemophilia setting. The comprehension of the IgG4 role in immune response may be crucial to establish the process for designing specific tolerance to FVIII.     

Effective and safe use of recombinant factor VIIa (NovoSeven®) in elderly mild haemophilia A patients with high-titre antibodies against factor VIII

Three patients with mild haemophilia A who developed high-titre antibodies against factor VIII at high age are reported. These patients had only a limited number of exposure days of FVIII concentrates in the past. The patients had to undergo surgery or presented with recurrent bleeding episodes. Treatment with recombinant FVIIa (rFVIIa) was effective and safe. Despite the high age and the presence of coronary heart disease in one of the patients, no adverse events or thrombotic complications occurred. These cases illustrate that the physician should always be alert on the development of inhibitors, also in elderly patients with mild haemophilia, in whom FVIII inhibitors had never been detectable before and that treatment with rFVIIa was effective and well-tolerated.     

High-titre factor VIII inhibitor in two children with mild haemophilia A

A frequently encountered complication of therapy given to patients with severe haemophilia A is the development of antibodies to infused factor VIII. While much less common, inhibitors also occur in patients with mild or moderate severity haemophilia A. Often thought to be of low titre and transient, several cases of high-titre inhibitors have been described in patients with mild or moderate haemophilia A. Generally these occur in adults or adolescents following significant infused factor VIII exposure. A review of reported cases revealed only two cases of high-titre inhibitor formation in mild haemophilia A patients younger than 10 years of age. We wish to report our experience with an additional two children with mild haemophilia A and high titre inhibitors, and offer suggestions for the management of these children.     

Prospective observational cohort studies for studying rare diseases: the European PedNet Haemophilia Registry

Haemophilia is a rare disease. To improve knowledge, prospective studies of large numbers of subjects are needed. To establish a large well‐documented birth cohort of patients with haemophilia enabling studies on early presentation, side effects and outcome of treatment. Twenty‐one haemophilia treatment centres have been collecting data on all children with haemophilia with FVIII/IX levels up to 25% born from 2000 onwards. Another eight centres collected data on severe haemophilia A only. At baseline, details on delivery and diagnosis, gene mutation, family history of haemophilia and inhibitors are collected. For the first 75 exposure days, date, reason, dose and product are recorded for each infusion. Clinically relevant inhibitors are defined as follows: at least two positive inhibitor titres and a FVIII/IX recovery <66% of expected. For inhibitor patients, results of all inhibitor‐ and recovery tests are collected. For continued treatment, data on bleeding, surgery, prophylaxis and clotting factor consumption are collected annually. Data are downloaded for analysis annually. In May 2013, a total of 1094 patients were included: 701 with severe, 146 with moderate and 247 with mild haemophilia. Gene defect data were available for 87.6% of patients with severe haemophilia A. The first analysis, performed in May 2011, lead to two landmark publications. The outcome of this large collaborative research confirms its value for the improvement of haemophilia care. High‐quality prospective observational cohorts form an ideal source to study natural history and treatment in rare diseases such as haemophilia.     

Mild haemophilia A discovered in a previously multi-operated 73-year-old man: characterization of a new mutation

A mild haemophilia A (factor VIII [FVIII] 10%) was discovered in a 73-year-old man during the preoperative haemostasis tests before a total knee arthroplasty for treatment of degenerative senile arthropathy. No history of previous abnormal bleeding was noted in spite of several previous challenging surgical procedures. Acquired haemophilia was ruled out and no other cases of haemophilia were found in the family. The surgery was successfully performed under recombinant FVIII aimed at achieving an FVIII level above 60%. This therapy induced the transient appearance of low-titre FVIII antibodies. A still unpublished mutation within the exon 3 (Gly73Ala) was found with molecular studies. This case report underlines the importance of haemostasis tests before high-risk bleeding surgical procedures even in elderly patients without a past history of haemorrhagic tendency.     

Retrospective audit of a continuous infusion protocol for haemophilia A at a single haemophilia treatment centre

Continuous infusion (CI) of factor concentrates has been suggested to decrease the risk of bleeding and reduce cost in the treatment of bleeding disorders. Concerns have also been raised regarding stability and sterility of products administered by CI, the risk of local thrombophlebitis and an association with the development of an inhibitor in mild haemophilia. A retrospective chart review was conducted to investigate a CI protocol regarding product use, maintenance of FVIII levels and the frequency of complications including inhibitor development. Twelve patients with haemophilia A received recombinant factor VIII by CI a total of 18 times between April 1998 and September 2003. Ages ranged from 4 months to 75 years and indications for treatment included severe bleeds and surgical prophylaxis. The protocol which was audited required a bolus of 50 U kg(-1) of FVIII followed by CI at an initial rate of 4 U kg(-1) hr(-1). All infusions were administered by i.v. infusion after diluting the reconstituted concentrate in saline. There were no documented cases of significant bleeding, adverse reactions, thrombophlebitis or infection. Two mild haemophilia A patients developed a low titre inhibitor after receiving CI. It is not clear in either case that CI was the main contributing factor. Our CI protocol will now be modified to use less product, aiming for more cost-effectiveness.     

Antibody formation and specificity in Bethesda‐negative brother pairs with haemophilia A

Antibodies directed towards non‐neutralizing epitopes on the factor VIII protein (FVIII) may be detected in patients with haemophilia A. We evaluated the prevalence of non‐neutralizing antibodies, in 201 inhibitor‐negative brother pairs with severe haemophilia A, enrolled in the Malmö International Brother Study and the Haemophilia Inhibitor Genetics Study. To evaluate binding specificity of the antibodies, ELISA plates were coated with two recombinant full‐length (FL) FVIII‐products and one recombinant B‐domain‐deleted (BDD) product. Seventy‐nine patients (39.3%) had a history of positive inhibitor titre measured by Bethesda assay, and FVIII antibodies were detected in 20 of them (25.3%). Additional 23 samples from subjects without a history of FVIII inhibitors were ELISA‐positive corresponding to a frequency of non‐neutralizing antibodies of 18.9%. The antibody response towards the different FVIII products was heterogenous, and was raised not only towards the non‐functional B‐domain but also towards both FL‐rFVIII and BDD‐rFVIII. In patients considered successfully treated with immune tolerance induction, 25.4% had remaining FVIII antibodies. The number of families with an antibody response in all siblings was increased when the total antibody response was taken into account, further supporting the concept of a genetic predisposition of the immune response. Further studies and careful monitoring over time are required to appreciate the immune response on the risk of inhibitor development or recurrence in the future.     

Rituximab for congenital haemophiliacs with inhibitors: a Canadian experience

When a high titre inhibitor develops in a patient with haemophilia, attempts are made to eradicate it through immune tolerance induction therapy (ITI) involving the frequent and regular administration of factor, usually for months to years. ITI is successful in only two thirds of patients prompting investigators to explore alternate regimens to use in haemophiliacs failing conventional ITI. Rituximab is an anti-CD20 monoclonal antibody, which has shown promise in the treatment of B-cell-mediated disorders. We developed a protocol for the use of rituximab in haemophilia A (HA) patients failing conventional ITI or in those haemophiliacs where the likelihood of success of conventional ITI is poor. Patients receive 375 mg m(-2) of intravenous rituximab weekly for 4 weeks followed by monthly (up to 5 months) until inhibitor disappearance and establishment of normal FVIII pharmacokinetics (recovery and half-life). Patients are concurrently placed on recombinant FVIII (100 U kg(-1) day(-1)). We have placed five haemophiliacs (four children with severe HA, and one adult with mild HA) on this protocol. In three patients (two with severe HA and one with mild HA) inhibitors disappeared although in neither severe haemophiliac did FVIII pharmacokinetics completely normalize. The fourth patient had a significant drop in inhibitor titres although not a complete disappearance of the inhibitor. All four of these patients ceased bleeding following rituximab. The fifth patient had no response to rituximab. This non-responding patient was not placed on concurrent FVIII. Our five cases suggest that rituximab may hold promise in the eradication of inhibitors. Prospective randomized studies are required to determine the value of this agent in inhibitor management.     

Assay discrepancy in mild haemophilia A: entire population study in a National Haemophilia Centre

Summary.  Assay discrepancy in mild haemophilia, here defined by a significantly higher factor VIII (FVIII):C response by the one-stage procoagulant assay as compared with a two-stage enzymatic method, has repeatedly been reported in literature. The purpose of this study was to determine the overall prevalence of this phenomenon amongst mild haemophilia families from a population of 2.95 million inhabitants in the Western Danish region. Information was collected retrospectively through a thorough search of archives of the National Haemophilia Centre in Aarhus. We identified 109 patients with mild haemophilia A amongst whom 92 were eligible to enter the study. These represent a total of 53 unrelated families. Our data illustrate that this assay discrepancy pattern is found quite frequently amongst our mild haemophilia A families. While the ratio of FVIII:C chromogenic/FVIII:C clot values was quite consistent amongst patients belonging to same family pattern, ratios in the entire cohort of families ranged from 0.18 to 1.00. Selecting a cut-off level for the FVIII:C chromogenic/FVIII:C clot ratios at 0.7, 0.6 and 0.5, respectively, we found that 38 (72%), 27 (51%) and 19 (36%) of families, respectively, displayed this assay discrepancy. In 10 patients, the FVIII:C chromogenic level was inside the category of moderate haemophilia at >0.01–<0.05 IU mL⁻¹, pointing to a class-shift in the biochemical phenotype. In conclusion, our data illustrate a substantial prevalence of the assay discrepancy phenomenon amongst mild haemophilia A patients in our geographical area.     

Analysis of low frequency bleeding data: the association of joint bleeds according to baseline FVIII activity levels

Summary. Many studies in the field of haemophilia and other coagulation deficiencies require analyses of bleeding frequencies. In haemophilia, the association of bleeding frequency with factor VIII (FVIII) activity levels is known from experience, but significant results are lacking. Bleeding frequencies in haemophilia are highly skewed count data, with large proportions of zeros. Both the skewness and the high amount of zeros pose a problem for standard (linear) modelling techniques. This study investigated the optimal analysing strategy for bleeding data by using the association of residual clotting factor level and number of joint bleeds in moderate and mild patients treated on demand as example. In total, 433 patients with moderate (27%) and mild (73%) haemophilia A treated on demand were included in this study. One year of self‐reported data on joint bleed frequency and baseline clotting factor activity were analysed using Poisson, negative binomial, zero‐inflated Poisson, and zero‐inflated negative binomial distributions. Multivariate regression analysis using negative binomial distribution provided the optimum data analytical strategy. This model showed 18% reduction [Rate ratio (RR) 0.82; 95%confidence interval (CI) 0.77–0.86] of bleeding frequency with every IU dL^‐1 increase in residual FVIII activity. The actual association is expected to be higher because of exclusion (30 out of 463 patients) of patients on prophylaxis (baseline FVIII levels 0.01–0.06 IU mL^?1). The best way to analyse low frequency bleeding data is using a negative binomial distribution.     

Immune response against serial infusion of factor VIII antigen through an implantable venous-access device system in haemophilia A mice

Haemophilia A is a life long bleeding disorder caused by an inherited deficiency of factor VIII (FVIII). About 30% of haemophilia A patients develop neutralizing antibodies as a consequence of treatment with FVIII concentrates. Immune tolerance protocols for the eradication of inhibitors require daily delivery of intravenous FVIII. We evaluated the immune responses to serial intravenous administration of FVIII in preimmunized haemophilia A mice. We introduced an implantable venous-access device (iVAD) system into haemophilia A mice to facilitate sequential infusion of FVIII. After preimmunization with FVIII, the haemophilia A mice were subjected to serial intravenous administration of FVIII through the iVAD system. In all mice with serial infusion of FVIII, high titers of anti-FVIII inhibitory antibodies developed at 10 exposure days (EDs). However, the anti-FVIII IgG titers were decreased after 150 EDs of sequential low-dose infusion of FVIII [0.05 U g(-1) body weight (BW) five times per week]. Proliferative response to ex vivo FVIII stimulation was significantly suppressed in splenic CD4(+) T cells from mice with serial low-dose FVIII infusion compared with those from mice with high-dose FVIII infusion (0.5 U g(-1) BW five times per week) or preimmunized mice. Moreover, splenic CD4(+) T cells from mice with serial low-dose infusion of FVIII failed to produce interleukin-2 and interferon-γ. These data suggest that serial infusion of FVIII could induce T-cell anergy in haemophilia A mice with inhibitor antibodies.     

Advances in knowledge of inhibitor formation in severe haemophilia A

Anti-drug antibody formation following factor VIII (FVIII) replacement therapy is the most important treatment-related complication in patients with severe haemophilia A. A significant number of these antibodies show neutralising activity against FVIII and are referred to as FVIII inhibitors. Alloimmunity to FVIII, given the absence of endogenous circulating FVIII protein, may be predictable to some extent; however, only 30% of patients develop inhibitors. Genetic and environmental risk factors have been identified, contributing to the likelihood of inhibitor development. Multiple immunological theories have been proposed which in part explain the outcomes of many epidemiological studies. Significant differences exist among replacement therapies, including the source, FVIII sequence, glycosylation, formulation components, impurities and aggregation potential, which significantly complicate interpretation of the results from these studies. In this review, we present recent advances in the understanding of the cellular mechanisms of inhibitor formation and highlight some areas of uncertainty requiring further investigation.     

Potential role of the dynamic properties of whole blood coagulation in assessment of dosage requirements in haemophilia

In the clinical setting, patients suffering from haemophilia are classified according to the residual level of the deficient coagulation factor. Patients suffering from the severe form of haemophilia (critical factor level <0.01 IU mL-1) display some heterogeneity in their tendency to bleeding despite the uniform factor level. Utilizing a new thrombelastographic method in which coagulation is activated by very small amounts of tissue factor and where resulting data are processed with new software, we studied the whole blood coagulation profile in 11 patients with severe haemophilia A and 11 patients with moderate haemophilia. In both groups of patients, we found a considerable degree of heterogeneity in the coagulation signal. In moderate haemophilia with factor VIII (FVIII):C levels between 0.01 and 0.05 IU mL-1, variance was expected, whereas a quite substantial diversity had not been forecasted in patterns of the whole blood coagulation profiles in patients with the severe form of haemophilia A. Ex vivo substitution to patient's blood to reach various theoretical levels of recombinant factor VIII (rFVIII) revealed that the coagulation response to FVIII supplementation varied substantially. In some severely affected patients levels of FVIII:C close to 0.05 IU mL-1 was sufficient to normalize the coagulation profile, while others required a dose giving >0.50 IU mL-1 of FVIII to achieve a normal whole blood clotting profile. In conclusion, our study revealed that severe haemophilia A seems not to be a single entity, but rather several different clinical and biochemical phenotypes, and that the response to added FVIII varies amongst patients.     

High incidence of anti-FVIII antibodies against non-coagulant epitopes in haemophilia A patients: a possible role for the half-life of transfused FVIII

The occurrence of antibodies (Abs) capable of inhibiting factor VIII (FVIII) coagulant activity is a severe complication in haemophilia A, leading to the inhibition of transfused FVIII activity. It is not known whether, or to what extent, post-transfusion antibodies may also arise against non-coagulant epitopes. Therefore we set up a system capable, in theory, to detect all the FVIII-induced antibodies by use of an enzyme-linked immunoassorbent assay (ELISA) based on coating human recombinant FVIII onto polystyrene microtitre plates. Serum samples from 23 patients affected by haemophilia A of different gravity (22 referred to our Centre and one to the Bari Centre) were analysed. Although only one patient was positive at Bethesda assay, the presence of antibodies in ELISA was detected in 39% of patients in variable degrees; transfusion with FVIII was found to induce a raise in antibody titre, arguing in favour of the specificity of the phenomenon. The clinical relevance of these non-inhibitory antibodies was evaluated in three patients; although half-life did not show any change in the patients without or with low amount of antibodies, FVIII clearance was found enhanced in the patient displaying high titre antibodies. We propose detection of anti-FVIII antibodies by ELISA when routinely assessing haemophilia A patients.