Evaluation of three automated chromogenic FVIII kits for the diagnosis of mild discrepant haemophilia A

In some mild haemophilia A patients (discrepant phenotype), coagulation FVIII levels by one-stage assay (FVIII-1st) are more than double those by classical two-stage coagulation assay (FVIII-2st), and may fall within the normal range. Our aim was to assess automated two-stage chromogenic FVIII assays (FVIII-chr) for diagnosis of mild discrepant haemophilia A. Three chromogenic FVIII kits (Biophen, Coamatic and Dade-Behring) were evaluated, using recommended and extended incubation times. Samples were tested from patients with discrepant haemophilia (n = 7) and equivalent mild haemophilia (agreement between FVIII-1st and FVIII-2st, n = 4). For equivalent haemophilia, FVIII-chr were consistent with FVIII-1st and FVIII-2st for all kits at all incubation times. For discrepant haemophilia, using recommended incubation times, mean FVIII-chr using Biophen reagents was 22 IU/dl (range 13-31), with Coamatic 26 (17-34) and with Dade-Behring 41 (33-47), compared with 36 (27-44) for FVIII-1st and 8 (6-9) for FVIII-2st. FVIII-chr decreased as incubation time was increased with Biophen and Coamatic, but decreased less with Dade-Behring. FVIII-chr using the Dade-Behring kit gave similar results to FVIII-1st and is not suitable for diagnosis of mild discrepant haemophilia A. FVIII-chr by Biophen and Coamatic kits is suitable for diagnosis of these patients, especially with an extended incubation time.     

One‐stage and chromogenic FVIII:C assay discrepancy in mild haemophilia A and the relationship with the mutation and bleeding phenotype

Summary. The discrepancy of the levels of factor VIII activity (FVIII:C) by different assays in some mild and moderate haemophilic A patients has been long known. Specific mutations affecting FVIII:C discrepancies have been described. No consensus exit as to which method most accurately represents the FVIII cofactor function in vivo and which has a better correlation with the haemorrhagic clinical expression. We studied 163 mild A haemophiliacs, and detected discrepancies in 20% of the patients, most of whom presented higher levels of FVIII:C with the one‐stage assay. In nine families, the FVIII mutation was found, while three showed mutations not previously described (Leu1978Phe and Ser1791Pro associated with higher levels of FVIII:C by one‐stage method; Arg1639His in a patient with low level of FVIII:C by the one‐stage, but normal, chromogenic assay). Assessing the level of FVIII:C by different methods could help to learn the possible haemorrhagic expressions of patients.     

Cytokine profile and FVIII inhibitors development in haemophilia A

Haemophilia A is a hereditary bleeding disorder linked to the X chromosome characterized by a deficiency or defect in the coagulation factor VIII (FVIII). Individuals with this coagulopathy require constant infusions of FVIII to maintain their physical integrity and haemostasis. During treatment, some patients develop an immune response that produces antibodies to FVIII, also called inhibitors, affecting the pro‐coagulant activity of this protein. Despite the clinical relevance of FVIII inhibitors, the immune mechanisms that lead to their production are not known. This study investigated the immunological cytokine profile using plasma from HA patients which were either positive or negative for FVIII inhibitors and from healthy individuals. The results showed that healthy individuals and HA patients that do not develop FVIII inhibitors have a mixed immune response profile with high secretion of IFN‐γ, TNF‐α IL‐2 and IL‐5. In contrast, HA patients with FVIII inhibitors exhibited an anti‐inflammatory/regulatory immune response characterized by low levels of all measured cytokines except for IL‐4 and IL‐10. This profile may be related to the development and maintenance of the FVIII inhibitors. By comparing the cytokine profiles of the three different groups we have established a model explaining the immune activation resulting in the production of FVIII inhibitors in haemophilia A patients.     

DDAVP challenge tests in boys with mild/moderate haemophilia A

Desmopressin (DDAVP) increases plasma factor VIII coagulant activity (FVIII:C) levels in patients with mild/moderate haemophilia A. In some subjects, FVIII can be increased to haemostatic levels, thereby avoiding use of factor VIII concentrates. We reviewed our hospital's experience with 62 boys with FVIII:C levels 0.01-0.3 IU/ml who had a DDAVP challenge test (i.v. 0.3 microg/kg) following diagnosis. A therapeutic response was defined as a 1 h post-FVIII:C increase at least twofold over baseline and > 0.3 IU/ml. Of the total group, 29 (47%) boys responded to DDAVP, all of them with mild haemophilia (baseline FVIII:C > or = 0.05 IU/ml), yielding a response rate of 57% in this subgroup. Boys who responded to DDAVP had higher baseline FVIII:C levels (mean +/- SEM, 0.17 +/- 0.01 vs 0.10 +/- 0.01 IU/ml, P < 0.01) and were older (5.2 +/- 0.8 vs 3 +/- 0.4 years, P = 0.02) than those who failed to do so. The association between DDAVP response and age, however, remains unclear: seven boys who failed the initial challenge test responded to re-challenge after a mean of 6.3 years (median 4.9, range 0.5-12.5), increasing the response rate in boys with mild haemophilia to 71%. Age and FVIII:C association with DDAVP response are both important in boys with mild/moderate haemophilia A. Absence of response to DDAVP should therefore be confirmed by later re-challenge.     

Functional characteristics of N8, a new recombinant FVIII

Summary. The aim of this study was to evaluate the in vitro function of the new recombinant factor VIII (FVIII) compound, N8. The specific activity of N8 as measured in a FVIII:C one‐stage clot assay was 9300 ± 400 IU mg^?1 based on the analysis of seven individual batches. The ratio between the FVIII:C activity measured in clot and chromogenic assays was 1.00 (95% confidence interval 0.97–1.03). N8 bound to von Willebrand factor with K_d values of 0.2 nm when measured by ELISA and by surface plasmon resonance. FVIIIa cofactor activity was determined from the kinetic parameters of factor IXa‐catalysed factor X (FX) activation. The rate of activation of N8 by thrombin as well as K_m and k_cat for FX activation was in the same range as those observed for Advate^®. The rate of activated protein C (APC)‐catalysed inactivation was similar for activated N8 and Advate^®. N8 improved thrombin generation in a dose‐dependent manner and induced similar rates of thrombin generation as Advate^® and the plasma‐derived FVIII product Haemate^®. Using thromboelastography (TEG^®), N8 was shown to improve the clot formation and clot stability in whole blood from haemophilia A patients. Comparable potency and efficacy of N8 and Advate^® was found based on TEG^® parameters. Finally, similar binding profiles to immobilized lipoprotein receptor‐related protein (LRP) of N8 and Advate^® were observed. The study demonstrated that N8 is fully functional in a variety of assays measuring FVIII activity. No functional differences were found between N8 and comparator compounds.     

Tyr2105Cys mutation in exon 22 of FVIII gene is a risk factor for the development of inhibitors in patients with mild/moderate haemophilia A

We report the case of a patient with mild haemophilia A, due to a Tyr2105Cys mutation in exon 22 of the C1 domain, who developed a high-titre factor VIII inhibitor (maximum titre 1600 BU) with recurrent severe haemorrhages and fatal intracranial bleeding. Based on published data, it appears that although this mutation occurs rarely in patients with mild or moderate haemophilia A, it is frequently associated with the development of high-titre inhibitors.     

Characteristics of inhibitors in mild/moderate haemophilia A

Summary.  Patients with mild or moderate haemophilia A usually have a mild bleeding disorder requiring only occasional treatment with factor VIII (FVIII) concentrates. The frequency of inhibitor development in such patients has been the subject of several recent surveys, which significantly modified our appreciation of this complication. Studies of the anti-FVIII antibodies provided an explanation for the different bleeding phenotypes observed in mild/moderate haemophilia A patients with inhibitors. Antibodies distinguishing between the patient's mutant FVIII and the normal wild-type FVIII were characterized, in addition to antibodies inhibiting completely or only partially FVIII activity. T lymphocytes recognizing FVIII and likely involved in the development of the immune response to FVIII were successfully identified. The FVIII peptides recognized by those FVIII-specific cells bind to many major histocompatibility complex (MHC) class II molecules, which may provide an explanation for the lack of strong association between MHC haplotypes and inhibitor development. Although these studies have advanced our understanding of the conditions leading to inhibitor development, further work is required to determine whether the mode of FVIII administration significantly influences inhibitor development. Further studies of the genetic factors are also required to fully understand the mechanisms leading to inhibitor development in patients with mild/moderate haemophilia A.     

Inhibitor development after switching of FVIII concentrate in multitransfused patients with severe haemophilia A

Switching between different therapeutic FVIII concentrate types has been postulated as a possible cause of inhibitor development in patient with haemophilia A. In this single‐centre, retrospective study, the incidence, titre and duration of inhibitor development in multitransfused patients, defined as patients with more than 150 exposure days (ED), were analysed from January 1970 to December 2007 in relation to ED and the number of switches between different products. Inhibitor titre was assessed by Bethesda assay (before 1998) or Nijmegen assay (after 1998). Medical records of 167 patients were screened, of which 97 patients met the inclusion criteria. Fourteen products of plasmatic origin (different purities) and five recombinant (three generations) were used. Nine patients (9%) developed inhibitors, all transient, low‐titre (1.41 ± 0.54 BU) after 323 ± 287 ED in average. Seventeen patients had no product switches of which four patients (23%) developed inhibitors (97 ED in average), whereas 13 patients (77%) did not (ED: 230). Fifty patients switched between plasmatic products only (median: 10 changes) of which five patients (10%) developed inhibitors (ED: 503), whereas 45 patients did not (ED: 932). Five patients switched between recombinant products only (seven changes) of which no patient developed inhibitors (748 ED). Twenty‐five patients switched between plasmatic and recombinant products (13 changes) of which no patient developed inhibitors (ED: 1654). No statistically significant differences between patient groups were observed. Neither the number of different FVIII products administered nor the switching of products influenced the incidence of inhibitor in multitransfused 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.     

Diagnosis and management challenges in patients with mild haemophilia A and discrepant FVIII measurements

Thirty per cent of patients with mild haemophilia A (MHA) present markedly different FVIII: C level when assayed by one‐stage clotting and two‐stage chromogenic assays. It is, therefore, a real clinical challenge to predict the individual bleeding risk of these patients. The aim of the present work was to study the relationship between the bleeding tendency of these patients with the results of a panel of phenotypic and genotypic tools. Thirty‐six patients with MHA were included in this multicentre prospective clinical study. The severity of bleeding symptoms was evaluated using the ISTH/SSC score. FVIII:C levels were measured using an activated partial thromboplastin time‐based one‐stage FVIII assay (FVIII: C1) and three commercial chromogenic kits (FVIII:CR). FVIII antigen levels, thrombin generation measurement and FVIII gene mutation analysis were also performed. Our results showed that a one‐stage FVIII: C assay cannot rule out the diagnosis of MHA, a combined use of FVIII:C1 with a FVIII:CR is suitable for detecting MHA. We observed that FVIII:CR results better reflected the clinical bleeding tendency of patients compared to FVIII:C1. We also observed a relationship between thrombin generation (TG) capacity and FVIII:CR of these patients. FVIII gene mutation analysis showed mutations previously reported in MHA patients with discrepant FVIII:C measurements, but with no predictive value of the individual bleeding phenotype of patients. Overall, we observed a relationship between chromogenic FVIII:C results, TG assay and bleeding tendency of patients with discrepant FVIII:C measurements, while FVIII:C1 was not well correlated with clinical bleeding phenotype in this particular population.     

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).     

Specific and global coagulation tests in patients with mild haemophilia A with a double mutation (Glu113Asp,Arg593Cys)

Background

Heterogeneous bleeding phenotypes are observed in haemophilia A patients with the same mutation in the F8 gene. Specific mutations in the A2 domain of factor VIII are associated with mild haemophilia and a higher risk of inhibitor development. Double mutations in mild haemophilia A are rarely reported. In this study, we investigated the in vitro function of factor VIII, performing different specific and global coagulation assays, observed clinical characteristics and assessed the possible predictive diagnostic value of the differences.

Materials and methods

The clinical features of haemophiliacs with a mild phenotype were reviewed. Blood samples were obtained and analysed for mutations and coagulation assays: activated partial thromboplastin time, one-stage and chromogenic factor VIII activity, factor VIII antigen and rotational thromboelastometry.

Results

We report on a cohort of 22 patients with double Glu113Asp, Arg593Cys mutations. All our patients have a quantitative defect of factor VIII and preserved similar functional activity. Factor VIII activities measured by the one-stage or chromogenic method were not discrepant, although the chromogenic assay resulted in 20% lower factor VIII activities. Waveform analysis showed a lower maximum value of the second derivative curve (Max2) of APTT with curve shape alternation, while thromboelastometry (INTEM) showed low sensitivity in comparison to results in a normal population.

Discussion

In genotyping, the coexistence of a second mutation should never be excluded, especially in cases of discordant clinical presentation. Waveform analysis correlates better with factor VIII activity than thromboelastometry and the Max2 parameter could provide additional information in managing haemophilia patients. The utility of specific factor activity and global haemostatic assays in general practice still needs to be investigated.     

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.     

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.     

Immunoglobulin isotypes and functional anti‐FVIII antibodies in response to FVIII treatment in Balb/c and C57BL/6 haemophilia A mice

Summary. Previous studies have demonstrated that genetic factors play an important role in determining the likelihood of formation of anti‐factor VIII (FVIII) antibodies in haemophilia A patients. We were interested in characterizing the spectrum of FVIII antibody formation and the primary and secondary immune responses after FVIII administration in two different exon 16‐disrupted haemophilia A mouse strains, Balb/c and C57BL/6. Balb/c and C57BL/6 E16 haemophilia A mice were used in all experiments. Total FVIII antibodies and FVIII inhibitors were measured using ELISA and Bethesda assays respectively. T‐ and B‐cell cytokines were quantified using ELISA and flow cytometry. FVIII antibodies, but not functional inhibitors were detectable 1 week after the first FVIII treatment in both strains. These antibodies mainly belonged to the IgM and IgA isotypes. After the fourth FVIII treatment, neutralizing anti‐FVIII antibodies were detected in both mouse strains: Balb/c (mean inhibitory titer 58 BU) and C57BL/6 (mean inhibitory titer 82 BU). IgG1 levels were similar in both strains but the IgG2A and IgG2B subclasses were higher in C57BL/6 mice. The results of intracellular cytokine staining of T cells indicated that the FVIII‐treated C57BL/6 mice produced more IL10 and Th1 cytokines than the FVIII‐treated Balb/c mice. These studies show that C57BL/6 mice develop a stronger immune response towards FVIII than Balb/c mice. We propose that the enhanced Th1 and IL10 cytokine micro‐environment induced in C57BL/6 mice is responsible for this difference. Therefore, genetic strain‐dependent differences must be considered when evaluating immunological outcomes in mouse models of haemophilia A.     

DDAVP responsiveness in children with mild or moderate haemophilia A correlates with age,endogenous FVIII:C level and with haemophilic genotype

Summary. In most individuals with moderate/mild haemophilia A, FVIII:C levels increase following DDAVP administration to a haemostatic range, thus avoiding the need for FVIII concentrates. We sought to determine the relationship between responsiveness to DDAVP in boys (<18 years old) with mild/moderate haemophilia and patient age, haemophilic severity and haemophilic genotype. Our cohort consisted of 13 boys with moderate and 61 boys with mild haemophilia who, between them, had 38 different mutations; 21 had unique mutations not shared by any other clinic patient, whereas 53 shared one of 17 mutations with some other clinic patient (included 26 boys with ≥1 haemophilic brother). Patient age and endogenous FVIII:C levels were strong predictors of response to DDAVP. Younger patients responded less well to DDAVP and 10 of the 11 patients, when retested at an older age, showed an improved response to DDAVP. Only 1 patient with moderate haemophilia responded to DDAVP, whereas 80% of patients with mild haemophilia responded (including all patients with an endogenous FVIII:C of >0.15 U mL⁻¹). Almost all patients with the same mutation had the same response to DDAVP or only a minor discordance in response. Patient’s age, disease severity and genotype all are predictors of response to DDAVP.     

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.     

Normalization of factor VIII levels in a patient with mild haemophilia A during a 35-year period

We report the case of a 45-year-old man who was diagnosed in June 1969 9 years old as having mild haemophilia A following a traumatic left shoulder bleed when his factor VIII (FVIII) activity was 11 IU dL(-1) based on a two-stage assay. The bleed resolved following treatment with intravenous cryoprecipitate. There was no family history of haemophilia. Cryoprecipitate infusions were required to treat further traumatic bleeds between 1971 and 1981. During this time, his FVIII activity was confirmed at 14 IU dL(-1). He defaulted many hospital appointments until 1991 when his FVIII activity had risen to 42 IU dL(-1). There was no evidence of infection, inflammatory or liver disease to account for this change. By 2005 he had a normal FVIII activity of 62 IU dL(-1) based on a one-stage assay. FVIII gene analysis confirmed a codon 531 mutation. It appeared that the discrepant FVIII results related to whether a two-stage or one-stage assay was used that has been previously reported for other patients with these mutations. We felt it important to raise awareness that this phenomenon may lead to apparent correction of haemophilia A.     

Parameters influencing FVIII pharmacokinetics in patients with severe and moderate haemophilia A

In haemophilia A patients factor VIII (FVIII) recovery and half‐life can vary substantially. There are parameters known to modulate FVIII pharmacokinetics (PK), but they explain only about 34% of the variability. The aim of this study was to identify new parameters that influence FVIII PK and thus to expand the current knowledge. FVIII PK were determined in 42 haemophilia A patients (37 severe, 5 moderate) without inhibitor. Patients' characteristics and laboratory parameters were evaluated for an association with FVIII PK. We analysed plasma levels of low‐density lipoprotein receptor‐related protein 1 (LRP1) and protein C (PC) activity, which had been hypothesized to influence FVIII activity. Furthermore, four variations in intron 6 of the LRP1 gene, which had been shown to influence LRP1, were investigated. FVIII half‐life differed widely from 6.2 to 20.7 h, with a median of 10.0 h. Patients with blood group O had shorter FVIII half‐life compared to patients with non‐O blood group (median FVIII half‐life 9.0 h vs. 10.4 h, P = 0.018). Age was significantly associated with FVIII half‐life (r = 0.32, P = 0.035). Besides age, also VWF antigen (r = 0.52, P < 0.001) and blood group (r = ?0.37, P = 0.015) was associated with FVIII half‐life. No correlation was found with FVIII‐ or LRP1‐genotype, LRP1 or PC concentrations. Our data showed large differences in FVIII PK between individual patients and revealed age, blood group and VWF levels as important determining factors for FVIII half‐life. FVIII genotype or levels of LRP1 or PC had no influence on FVIII PK.     

Inhibitors in haemophilia: pathophysiology

Summary.  Development of inhibitors to coagulation factors is one of the major problems faced by people with haemophilia. Up to a third of patients, following treatment with factor concentrates, will develop an antibody (inhibitor) to that factor, rendering it inactive, and leaving the patient at risk from life-threatening bleeding. Evidence shows that this immune response is T-cell-dependent, but as yet, the epitopes responsible have not been identified. Risk for inhibitor development is highest within the first 50 days of treatment, with reactions being rare after 200 days. The risk is mediated by the major histocompatibility complex class of the patient, and by mutations in the factor VIII genotype, with large deletions conferring greatest risk.