Hemophilia pathophysiology

Hemophilia Microchapters
Home
Patient Information
Overview
Historical Perspective
Classification
Pathophysiology
Causes
Differentiating Hemophilia from other Diseases
Epidemiology and Demographics
Risk Factors
Screening
Natural History, Complications and Prognosis
Diagnosis
Diagnostic Study of Choice
History and Symptoms
Physical Examination
Laboratory Findings
Electrocardiogram
X-ray
Echocardiography and Ultrasound
CT scan
MRI
Other Imaging Findings
Other Diagnostic Studies
Treatment
Medical Therapy
Surgery
Primary Prevention
Secondary Prevention
Cost-Effectiveness of Therapy
Future or Investigational Therapies
Case Studies
Case #1
Hemophilia pathophysiology On the Web
Most recent articles
Most cited articles
Review articles
CME Programs
Powerpoint slides
Images
American Roentgen Ray Society Images of Hemophilia pathophysiology All Images X-rays Echo & Ultrasound CT Images MRI
Ongoing Trials at Clinical Trials.gov
US National Guidelines Clearinghouse
NICE Guidance
FDA on Hemophilia pathophysiology
CDC on Hemophilia pathophysiology
Hemophilia pathophysiology in the news
Blogs on Hemophilia pathophysiology
Directions to Hospitals Treating Hemophilia
Risk calculators and risk factors for Hemophilia pathophysiology

Editor-In-Chief: C. Michael Gibson, M.S., M.D. [1];Associate Editor(s)-in-Chief: Simrat Sarai, M.D. [2]

Overview

Hemophilia is a genetic bleeding disorder resulting from the insufficient levels of clotting factors in the body. The clotting factors irregularity causes a lack of clumping of blood required to form a clot to plug a site of a wound. The genes involved in the pathogenesis of hemophilia include the F8 gene in hemophilia A and F9 gene in hemophilia B and C. Hemophilia predominantly affects the male population but the sub-type hemophilia C, with an autosomal inheritance pattern, can affect the males as well as females.

Pathophysiology

• Hemophilia is caused by a mutation or change, in one of the genes, that provides instructions for making the clotting factor proteins needed to form a blood clot. This change or mutation can prevent the clotting protein from working properly or to be missing altogether. These genes are located on the X chromosome. Males have one X and one Y chromosome (XY) and females have two X chromosomes (XX). Males inherit the X chromosome from their mothers and the Y chromosome from their fathers. Females inherit one X chromosome from each parent.

• The X chromosome contains many genes that are not present on the Y chromosome. This means that males only have one copy of most of the genes on the X chromosome, whereas females have 2 copies. Thus, males can have a disease like hemophilia if they inherit an affected X chromosome that has a mutation in either the factor VIII or factor IX gene. Females can also have hemophilia, but this is much rarer. In such cases both X chromosomes are affected or one is affected and the other is missing or inactive. In these females, bleeding symptoms may be similar to males with hemophilia.

• A female with one affected X chromosome is a "carrier" of hemophilia. Sometimes a female who is a carrier can have symptoms of hemophilia. In addition, she can pass the affected X chromosome with the clotting factor gene mutation on to her children.

• Even though hemophilia runs in families, some families have no prior history of family members with hemophilia. Sometimes, there are carrier females in the family, but no affected boys, just by chance. However, about one-third of the time, the baby with hemophilia is the first one in the family to be affected with a mutation in the gene for the clotting factor.

Hemophilia – The hemophilias are bleeding disorders caused by deficiency of clotting factors:

•Deficiency of factor VIII (factor 8) – Hemophilia A

•Deficiency of factor IX (factor 9) – Hemophilia B

Hemophilia A and B severity is classified according to the level of circulating factor activity, which correlates with bleeding risk and the risk of inhibitor development. Mild disease is defined as a factor activity level above 5 and below 40 percent; moderate disease is a factor activity level between 1 and 5 percent; and severe disease is a factor activity level below 1 percent [1]. (See "Clinical manifestations and diagnosis of hemophilia", section on 'Definitions'.)

Hemophilia C is sometimes used to refer to factor XI (factor 11) deficiency, but the phenotypic disease severity, type of bleeding manifestations, and management differ from hemophilia A and B. (See "Factor XI (eleven) deficiency".)

Acquired hemophilia refers to development of a clotting factor deficiency that was not present at birth. Typically, this occurs when an autoantibody is produced against an endogenous factor. Acquired hemophilia A (acquired inhibitor against endogenous factor VIII) is the most common. (See "Acquired inhibitors of coagulation".)

●Treatment – Previously untreated patients (PUPs) are those who have not received infusions of replacement factor. Previously treated patients (PTPs) are those who have been exposed to replacement factor.

●Exposure days – Exposure days refers to the number of days on which the individual was exposed to exogenous factor. As an example, an individual treated with factor infusions on Monday, Wednesday, and Friday for two weeks has six exposure days. An individual treated with two infusions of factor on Monday has one exposure day. The number of exposure days correlates with the risk of inhibitor development, but it is not necessarily a good surrogate for treatment intensity, as discussed below. (See 'Therapy considerations' below.)

●Inhibitors – An inhibitor is a neutralizing antibody that interferes with the function of the factor against which it is directed. In hemophilia A and B, inhibitors are alloantibodies directed against infused factor. Not all antibodies to factor act as inhibitors. Non-neutralizing antibodies directed against coagulation factors may be seen in individuals with hemophilia and in the general population [2].

•Titer – Inhibitor titers are quantified using the Bethesda assay. This is a clot-based assay in which serial dilutions of patient plasma are incubated with pooled normal plasma and residual clotting activity is measured. In many cases, the Nijmegen modification is used. Heat treatment is sometimes used to remove any exogenous factor VIII and more accurately determine the patient's true inhibitor titer. Individuals receiving emicizumab cannot have an inhibitor titer determined using a clot-based assay. A chromogenic Bethesda assay (CBA) is used for individuals receiving emicizumab.

The titer is the reciprocal of the dilution of patient plasma that results in 50 percent factor activity, expressed as Bethesda units (BU) or Nijmegen-Bethesda units (NBU). The titer may also be expressed in BU/mL. The greater the dilution required to titrate out the inhibitor, the higher the inhibitor titer.

•High- versus low-responding inhibitor – An inhibitor titer of ≥5 BU at any time is considered a high-responding inhibitor, and a persistent titer <5 BU despite repeated factor VIII exposure is considered a low-responding inhibitor [3,4]. Low-responding inhibitors do not rise to ≥5 BU with repeated exposure to factor. A patient whose titer is ≥5 BU has a greater likelihood of increase upon re-exposure to factor.

•Transient inhibitors – Inhibitors that disappear spontaneously (ie, decrease below the definition threshold within six months of initial documentation despite continuing exposure to factor) are referred to as transient inhibitors [1].

●Immune tolerance induction – Immune tolerance induction (ITI, also called immune tolerance therapy [ITT] or inhibitor eradication) is the primary method used to eliminate or control inhibitors. It involves administration of frequent, regularly scheduled doses of the deficient factor to reset (tolerize) the individual's immune system and reduce production of the antibody. The immunologic principles are somewhat similar to immunotherapy for allergic disease, although there are significant differences in the clinical manifestations of the immune response. (See "Allergen immunotherapy for allergic disease: Therapeutic mechanisms" and 'Immune tolerance induction' below.)

PATHOGENESIS

Mechanisms of formation and action — Inhibitors form when the immune system recognizes the infused factor as foreign and generates neutralizing antibodies. Predisposing and provoking factors are enumerated below. (See 'Predisposing influences' below.)

Inhibitors are high-affinity antibodies (primarily immunoglobulin G [IgG]) directed against the factor protein [5]. They are often polyclonal (ie, separate antibody specificities produced by separate B cell clones that recognize distinct epitopes). In contrast, inhibitors in individuals with acquired hemophilia are often monoclonal. In one study, approximately 80 percent of individuals with hemophilia A who developed inhibitors had at least two or more independent antibody specificities against factor VIII [6].

In hemophilia A, most antibodies are directed against the A2 and/or C2 domains of the protein. Mechanisms include the following:

●Interference with factor VIII binding to phospholipids or von Willebrand factor (VWF) via binding to the C2 domain [6-13]

●Interference with factor VIII binding to factor IX or blocking of the intrinsic ten-ase activity of the factor VIIIa-factor IXa complex [14,15]

●Increased clearance of factor VIII via direct proteolysis [16,17]

The domain structure of the factor VIII and factor IX molecules and functional regions of the proteins are discussed separately. (See "Biology and normal function of factor VIII and factor IX".)

Predisposing influences — Influences that contribute to inhibitor development are complex, multifactorial, and incompletely understood [3]. They are likely to involve characteristics of the patient (eg, variations in factor genes and immune regulatory genes, race), the environment (eg, surgery, trauma, or other danger signals), and the hemophilia treatment (eg, age at first exposure, product used, intensity of dosing, use of prophylaxis versus on-demand treatment).

Sometimes these elements may be difficult to tier in importance because most of the available data come from observational studies that may be subject to a variety of interrelated and confounding factors. As examples:

●Different populations in different parts of the world may have different factor and immune response genotypes and may be treated using different products at different intensities, starting at different ages

●Individuals with more severe disease are likely to have more exposure days earlier in their disease course

●Individuals in certain populations may co-inherit certain factor mutations and certain mutations affecting the immune response

Risk factors for inhibitor development in hemophilia A have been studied extensively compared with hemophilia B because hemophilia A is more common and the incidence of inhibitors is higher. Further, hemophilia B is often associated with point mutations, which are less commonly associated with inhibitor development, rather than deletions. The extent to which the information on predisposing factors can be generalized to hemophilia B is not known and may differ substantially. Research continues to define the best predictors of inhibitor development in both hemophilia A and B, as well as methods to decrease or prevent formation.

Patient characteristics — Inhibitors are much more common in hemophilia A than in hemophilia B. (See 'Epidemiology' below.)

Beyond this, there are several established patient characteristics that increase inhibitor risk. These include genetic factors related to the specific factor mutation and those related to immune response genes. Race and ethnicity also play a role, but their mechanisms are not well-elucidated.

●Factor variant – The factor mutation (variant) determines disease severity, and more severe disease is associated with greater inhibitor risk. This is likely because lower levels (or absence) of endogenous factor will make it less likely that the individual's immune system was tolerized to the antigen during development and more likely that the infused product is recognized by the recipient's immune system as foreign. There may also be factor antigen circulating in some individuals with absent factor activity, and these individuals may be more likely to be tolerized to that factor.

Thus, large deletions and nonsense mutations (creation of an internal stop codon) in the F8 gene are much more likely to be associated with inhibitors compared with small deletions, insertions, or missense mutations (creation of an amino acid change) [3,18-22]. The intron 22 gene inversion, present in 40 to 45 percent of individuals with severe hemophilia A, carries an intermediate risk [21,23]. (See "Genetics of hemophilia A and B", section on 'Factor VIII (F8) gene/hemophilia A'.)

•In a meta-analysis of studies that examined F8 genotype and inhibitor risk in 5383 individuals with hemophilia A using intron 22 inversions as the control group, the odds ratios (ORs) for inhibitor development with large deletions, nonsense mutations, small deletions/insertions, and missense mutations were 3.6, 1.4, 0.5, and 0.3, respectively [23].

•In an analysis of 586 participants who had data entered in the Haemophilia A Mutation, Search, Test and Resource Site (HAMSTeRS registry), the frequencies of inhibitors were as follows [24]:

-Large (>200 bp) deletions – 38 percent

-Nonsense mutations – 36 percent

-Inversions – 20 percent

-Small (≤86 bp) deletions – 14 percent

-Missense mutations – 7 percent

Other studies have identified specific high-risk F8 variants in individuals with mild to moderate hemophilia A such as the C2 domain Trp2229Cys mutation and the Arg593Cys mutation [25,26].

As noted below, there are several normal F8 polymorphisms that do not cause hemophilia and may be distributed differently in individuals of different races; these may contribute to different rates of inhibitor development (eg, due to minor antigenic differences between endogenous factor VIII and the factor VIII protein sequence in the replacement product) [27]. (See 'Replacement product' below.)

Data are much more limited for hemophilia B, but they confirm a greater frequency of inhibitors in those with severe disease [28]. In a sequencing study, complete F9 gene deletions conferred a higher risk than truncating mutations, partial deletions, or missense mutations [29].

●Immune response genes – Development of inhibitors is an immune phenomenon, and some data have suggested that genes involved in the immune response to foreign proteins may contribute to inhibitor development [3]. As an example, the Hemophilia Inhibitor Genetics Study (HIGS) evaluated genes involved in immune regulation among 104 sibling pairs with hemophilia A who were discordant in inhibitor status [30]. As siblings, these individuals had the same F8 mutation. Analysis of single nucleotide polymorphisms (SNPs) demonstrated variations in 13 immune response/immune modifier genes that correlated with inhibitor development. Other studies have implicated specific genes including those that encode human leukocyte antigen (HLA) class II antigens, interleukins, tumor necrosis factor (TNF)-alpha, and/or cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) [31-36].

●Other genetic contributions – There may be other as yet undefined genetic factors that influence inhibitor development. This possibility is suggested by case-control studies that have found increased rates of inhibitors in individuals with a family history of inhibitors [19,25,37].

●Race – Individuals with African or Hispanic ancestry have a higher rate of inhibitor development compared with whites. A study from the Centers for Disease Control (CDC) in the United States that reported on the prevalence of inhibitors among 5651 people with hemophilia A found the following prevalence of inhibitors in different races [38]:

•Black – 27 percent

•Hispanic – 25 percent

•Non-Hispanic white – 16 percent

Other studies have reported significantly higher inhibitor prevalences in blacks and Asians with hemophilia A. As an example, a study involving 460 families in the Malmo international brother study (388 families with hemophilia A and 72 families with hemophilia B) reported inhibitors in 56 percent of blacks and 27 percent of whites [39]. The incidence of inhibitors in this study was as high as 50 percent in individuals with Asian, Indian, and Hispanic ancestry. In hemophilia B, a review from a large United States database (3800 patients) found that the OR for inhibitor development was 2.8 for blacks (95% CI 1.4-5.5) and 1.7 for Hispanics (95% CI 0.7-3.7) [28].

Race may be a surrogate for a number of biologic differences that affect the immune response and/or for differences in medical care.

As noted above, another connection between race and inhibitor development may involve mismatch between the amino acid sequence of the factor present in the replacement product and the endogenous factor in the recipient that could make the recipient's immune system more likely to recognize the replacement factor as foreign and generate an immune response. In a study that sequenced the F8 gene in 78 black people with hemophilia A who had developed inhibitors, the people with inhibitors were more likely to have endogenous factor VIII amino acid sequences that differed from the amino acid sequence in the recombinant factor VIII replacement products that were available at the time [27]. (See 'Replacement product' below.)

●Age – Age-associated changes in immune function may also affect inhibitor development. The risk is highest in children under the age of five years, and risk increases beyond the age of 60 years [40]. (See "Immune function in older adults".)

The correlation between age at which prophylaxis is initiated and risk of inhibitor development is complex, as discussed below. (See 'Therapy considerations' below.)

Many of these studies are affected by different frequency of inhibitor testing, making cross-study comparisons difficult.

Therapy considerations — Therapy-related factors that may influence inhibitor development in susceptible individuals include the source, purity, and formulation of the replacement product (eg, plasma-derived versus recombinant, full-length versus modified factor sequence, presence of von Willebrand factor [VWF] or other proteins) and the intensity of treatment (eg, dose, number of exposure days) [3]. The age at which factor replacement is initiated and switching products do not appear to have a major role in inhibitor development; however, the intensity of treatment, presence of danger signals, and use of prophylaxis may all influence the age at which treatment is started. (See 'Intensity of therapy (dose, schedule, number of exposure days)' below and 'External/environmental factors' below.)

Replacement product — There are a number of factor concentrates, including plasma-derived of various purities as well as recombinant products that contain various genetic sequences and/or sequence or other modifications to prolong half-life, and that are made with a number of different cell culture systems and purification protocols. Some plasma-derived products also contain VWF. No "best" factor replacement product that has all the optimal attributes for each patient. (See "Hemophilia A and B: Routine management including prophylaxis", section on 'Available products'.)

As each of the replacement products has become available at different times, and practices have varied over time and place, comparison with historical controls is especially difficult. There is only a single large randomized trial comparing different products, and no trial has compared all available products with each other.

The 2016 Survey of Inhibitors in Plasma-Product Exposed Toddlers (SIPPET) trial remains the only prospective randomized trial comparing replacement products. SIPPET randomly assigned 264 children under the age of six years with severe hemophilia A (factor VIII <1 percent) and no or minimal previous factor therapy to receive a recombinant factor VIII product or a plasma-derived factor VIII product containing VWF, and followed them for up to 50 exposure days or three years after randomization, whichever came first [41]. The treatment regimen (prophylaxis versus on-demand) was at the discretion of the treating clinician. The primary outcome was development of any inhibitor with a titer of at least 0.4 Bethesda units (BU) using a centrally performed assay (Nijmegen method). Secondary outcomes included development of high titer inhibitors (≥5 BU).The majority of patients were enrolled from India, Egypt, and Iran, with smaller numbers from the United States, Italy, and other countries.

The peak timing of inhibitor development in SIPPET participants was during the first 15 exposure days. Inhibitors developed in a higher percentage of the those treated with the recombinant factor product (47 of 126 individuals who received recombinant factor VIII [37 percent] versus 29 of 125 individuals who received plasma-derived factor VIII with VWF [23 percent]; hazard ratio [HR] for recombinant products 1.87) [41]. The results remained essentially the same when analyzed after removing data on second-generation products, which previously had been suggested to have an increased risk of inhibitor development. High titer inhibitors were also more frequent in the recombinant factor VIII arm (24 versus 16 percent).

There were two deaths, both in the plasma-derived factor VIII with VWF arm (one from bleeding and one motor vehicle accident) and nine episodes of severe bleeding (six in the recombinant factor VIII arm and five in the plasma-derived factor VIII with VWF arm). The rate of prophylaxis (rather than on-demand therapy) was slightly higher in the recombinant factor VIII arm (55 versus 51 percent).

There has been great interest in determining the generalizability and clinical application of the SIPPET results, as noted by the study's authors, and various post-hoc analyses of different patient groups in the trial appear to show consistency with the original trial results [42]. A more challenging question is whether the results can be generalized to other recombinant factor products (eg, third-generation or extended half-life products, versus the specific first-, second-, and third-generation recombinant products used in the trial) [43]. The following conclusions seem reasonable:

●For all patients, the choice of product is complex and must balance a number of benefits, risks, and burdens. For those who require treatment with a factor replacement product for prophylaxis or treatment of bleeding, we individualize the choice of product based on the patient's circumstances and needs, following an open discussion with the patient regarding data on specific products and patient values and preferences.

●All inhibitors in the SIPPET trial occurred before 39 exposure days, and all high titer inhibitors occurred before 34 exposure days. Thus, for individuals with hemophilia A who are receiving a recombinant factor VIII product, who have not developed an inhibitor, and who are past the high-risk exposure period, switching to a VWF-containing plasma-derived product is not indicated.

●Analysis of each specific recombinant product used in the trial did not alter the initial findings [42]. However, the trial did not include some of the recombinant products that have subsequently become available, which appear to have an inhibitor rate that may approach that seen with factor VIII plus VWF-containing products. Thus, the decision to avoid exposure to recombinant products during the early phases of treatment requires a discussion between the family and provider.

The Medical and Scientific Advisory Council (MASAC) of the National Hemophilia Foundation (NHF) in the United States and the European Medicines Agency have not advocated a change to plasma-derived products based on the results of the SIPPET trial [44-46]. (See 'Society guideline links' below.)

The mechanism of increased risk of inhibitors with certain recombinant products is unknown. The plasma-derived products studied in the SIPPET trial contained VWF and other plasma proteins and are likely to differ in post-translational modifications from the recombinant products used in the trial, which were manufactured from non-human cell lines. It is possible that VWF may mask epitopes on the infused plasma-derived factor VIII or may protect factor VIII from endocytosis by antigen-presenting cells [47]. However, not all plasma-derived products contain VWF, and some recombinant products are made in human cell lines; the impact of these differences on inhibitor development is unknown.

Further trials using the newer-generation recombinant products made in human cell lines and those with extended half-lives are ongoing.

Prior to the SIPPET trial, information on inhibitor development with recombinant products was obtained in the 2013 Research Of Determinants of INhibitor development among previously untreated patients with haemophilia (RODIN) study, which prospectively evaluated 574 children with severe hemophilia A [48]. Inhibitors developed in 32 percent (high titer inhibitors in 22 percent). The risk of inhibitor formation was higher with second-generation full-length recombinant products compared with third-generation recombinant products (adjusted HR 1.60; 95% CI 1.08-2.37); however, the implications of this finding were less clear due to lack of randomization and small numbers of patients.

A 2012 observational study of 111 patients with hemophilia A who developed inhibitors found that compared with those who received low/intermediate-purity factor VIII products, those who received high-purity plasma-derived products had an increased risk of inhibitor development (adjusted HR 2.0; 95% CI 1.1-4.0) and those who received recombinant factor VIII had a higher rate of inhibitor development (adjusted HR 4.9; 95% CI 2.9-8.3) [49]. A 2018 observational study of 395 patients with severe hemophilia A from France (the FranceCoag cohort) found that inhibitor development was more likely in those who received one of two recombinant products compared with a plasma-derived product (cumulative incidence at 75 exposure days, 20 or 32 percent versus 13 percent) [50].

Some observational studies published before the SIPPET trial found no difference between recombinant and plasma-derived products; however, these results may have been subject to confounding factors that were eliminated in the randomized trial [51,52].

Intensity of therapy (dose, schedule, number of exposure days) — The dose and schedule at which the product is administered is reflected in the number of exposure days (days on which the patient received one or more factor infusions) (see 'Terminology and definitions' above), which is a strong predictor of inhibitor development.

●Hemophilia A – Inhibitors typically develop during the first 20 exposure days, as illustrated in the following studies:

•Severe disease – In the SIPPET trial (randomized trial of recombinant versus plasma-derived factor VIII in 264 children with severe hemophilia A), all inhibitors developed before exposure day 39, and 90 percent occurred before exposure day 20; for high titer inhibitors, all occurred before exposure day 34 and 90 percent before exposure day 16 [53].

In the FranceCoag cohort (395 children with severe hemophilia A), the median number of exposure days at inhibitor detection was 14, and the greatest percentage of inhibitors developed before exposure day 25 [50].

•Mild to moderate disease – In the INSIGHT study, inhibitor development was assessed in 1112 people with mild to moderate hemophilia A (factor VIII levels 2 to 40 units/dL) who were treated with factor VIII, 59 of whom developed inhibitors [54]. Of these, 41 (69 percent) developed an inhibitor before 50 exposure days, 17 (29 percent) developed an inhibitor between 50 and 100 exposure days, and one developed an inhibitor after 100 exposure days. The cumulative risk of inhibitor development was calculated as 5 percent at 28 exposure days, 7 percent at 50 exposure days, and 13 percent at 100 exposure days.

●Hemophilia B – There are fewer data to use in determining the correlation between the number of exposure days and inhibitor development in individuals with hemophilia B. In a review of the 88 patients in the international hemophilia B database, those who developed inhibitors did so at a median of 11 exposure days [55].

However, dose intensity is not simply the number of exposure days. As an example, dosing twice daily for three consecutive days after a serious bleed is more intense than dosing once every other day for three days of prophylaxis, yet both are counted as three exposure days. Thus, dose intensity may constitute a separate consideration over and above the number of exposure days. Dose intensity may be a better surrogate than exposure days for factors that affect the immune response, such as the degree of tissue injury. (See 'Patient characteristics' above and 'External/environmental factors' below.)

If an individual requires factor administration for surgery or to treat bleeding, factor should not be withheld to reduce the risk of inhibitor development. Data are conflicting regarding whether the risk of inhibitors changes with continuous infusion versus intermittent dosing. Some studies have suggested that continuous infusion increases inhibitor rate and others have suggested the opposite [56,57]. Thus, we do not favor or disfavor continuous infusion; we use the therapy that is deemed optimal for hemostasis for the specific patient. (See "Treatment of bleeding and perioperative management in hemophilia A and B".)

Age at which factor prophylaxis is initiated — The age at which factor replacement is initiated is challenging to separate from the severity of disease. Starting prophylaxis early may reduce inhibitor development, especially if there is ongoing exposure without danger signals (eg, repeated exposures in the absence of surgery, trauma, or spontaneous bleeding). However, this must be balanced with the burdens of routine intravenous therapy in a young infant or child. This paradigm may change with the introduction of emicizumab for hemophilia A. (See "Hemophilia A and B: Routine management including prophylaxis", section on 'Age of initiation and dosing schedule'.)

Although not addressed in a randomized trial, the effect of age at first treatment seems to be a marker for other risk factors rather than an independent risk factor for inhibitor development. Two studies demonstrated that age at first treatment did not correlate with inhibitor development after adjustment for other factors:

●In a case-control study involving 108 children with hemophilia A, there was a trend towards greater likelihood of inhibitor development when factor was started at a younger age (<11 months versus ≥11 months), but this trend was no longer present after adjusting for genetic factors [19].

●In the Concerted Action on Neutralizing Antibodies in severe hemophilia A (CANAL) study, which retrospectively investigated 366 patients with hemophilia A, the rate of inhibitor development appeared to correlate with age at first replacement therapy, but the effect disappeared after adjustment for treatment intensity [20].

Switching products — There are multiple studies that address the role of switching products in inhibitor development; none of these support an increased risk of inhibitor development due to switching products in previously treated patients [58-60]. As an example, in an observational study in which all patients with hemophilia A in Ireland (113 individuals) were switched from one recombinant factor VIII product to another, only one developed a new inhibitor; this was a one-year-old boy who had only received three doses of the previous product and was therefore at high risk for inhibitor development [61].

If antibodies form in multiply-exposed patients who have been changed to a new product, it is reasonable to investigate product-related factors [58,62,63]. In this case, consideration should be given to the use of the prior product if the patient has had many exposure days in the past, although change to the prior product may not always result in inhibitor disappearance.

External/environmental factors — Some studies have suggested that the degree of injury or tissue damage present at the time of factor infusion may contribute to the immune response to the factor (ie, the "danger theory") [3,64]. More severe injury is likely to accompany major bleeding and/or surgery. Investigators in the RODIN study used the dose of factor VIII as a surrogate for more severe injury and analyzed the rate of inhibitor development relative to the factor VIII dose intensity in a subset of previously unexposed children given factor VIII [65]. High-intensity factor treatment was associated with an increased risk of inhibitor development (adjusted HR 2.0; 95% CI 1.3-3.0), while prophylactic therapy was associated with a lower risk (adjusted HR 0.61; 95% CI 0.35-1.1).

While these findings suggest a potential biologic mechanism, they should not be interpreted to advocate the use of less intensive treatment of severe bleeding. Bleeding is treated with factor infusions or bypassing therapy, as discussed separately. (See "Treatment of bleeding and perioperative management in hemophilia A and B", section on 'Acute therapy for bleeding'.)

Clinical trials are investigating the use of low-dose prophylactic factor infusions in the absence of danger signals as a means of reducing inhibitor development [66].

Several observational studies have found no correlation between administration of routine vaccinations (eg, influenza vaccine) and inhibitor development [19]. Preclinical studies suggest vaccinations may be associated with a lower risk of inhibitors [67].

Risk score (hemophilia A) — Data from a cohort of consecutive patients with severe hemophilia A in the CANAL study were used to develop a risk score for the development of factor VIII inhibitors based on three risk factors [68]:

●Family history of inhibitors – 2 points

●High-risk gene mutation – 2 points

●Intensive treatment at first bleeding episode – 3 points

In the initial study cohort (332 patients), inhibitor incidences for individuals with risk scores of 0, 2, or ≥3 points were 6, 23, and 57 percent, respectively. Similar incidences were noted in a validation cohort of 64 patients. Of note, this score requires that the patient be treated, a disadvantage when trying to predict the risk of inhibitor development in individuals who have not been exposed to exogenous factor VIII.

EPIDEMIOLOGYInhibitors are more common in hemophilia A than in hemophilia B, with overall prevalence in individuals with severe disease as follows:

●Hemophilia A – Approximately 20 to 30 percent [69-75]

●Hemophilia B – Approximately 1.5 to 3 percent [28,55,70,71,76]

The incidence of inhibitors is highest in individuals with severe disease during the first 50 factor exposure days, although individuals with mild to moderate disease can also develop inhibitors. It has been hypothesized that the presence of some circulating factor IX in most people with hemophilia B may result in a greater likelihood of immune tolerance and may partially explain the lower prevalence of inhibitors [55].

Certain groups such as blacks have higher inhibitor rates (up to 50 percent in those with severe hemophilia A and 5 percent of those with severe hemophilia B) [28,77]. (See 'Predisposing influences' above.)

TYPICAL PRESENTATIONIn some cases, especially in hemophilia A, an inhibitor may be clinically silent and may not lead to a marked increase in the frequency or severity of bleeding; inhibitors may be identified when the response to infused factor is inadequate (ie, when there is no increase in factor activity after factor infusion and/or bleeding does not cease with factor infusion). In a prospective surveillance study in the United States that enrolled 1163 individuals with hemophilia A or B, 23 new factor VIII inhibitors were identified, and of these, 14 (61 percent) were clinically silent [78]. These data underscore the need for ongoing routine inhibitor surveillance in hemophilia. (See 'Routine screening and preoperative testing' below.)

However, a patient receiving prophylactic factor infusions who develops an inhibitor may experience new breakthrough bleeding or may have poor or delayed resolution of bleeding. Patients with inhibitors may also have altered pharmacokinetics of the replacement factor, which may result in impaired hemostasis and an increased rate of musculoskeletal complications. Over time, inhibitors alter the disease course, as affected individuals have poorly controlled joint bleeding and develop target joints, with subsequent increased bleeding in these joints due to acute and chronic synovitis [79]. An inhibitor should be suspected when any bleeding episode is refractory to usual therapy, particularly in patients with severe hemophilia.

Individuals with mild or moderate hemophilia (factor activity between 1 and 40 percent) who develop inhibitors that crossreact with their endogenous factor may be converted to a more severe state. One series identified factor VIII inhibitors in 26 patients with mild or moderate hemophilia A; most of the inhibitors developed after a period of intensive replacement therapy [25]. Spontaneous bleeding occurred in 22, with a bleeding pattern similar to that of acquired hemophilia in 17.

High titer inhibitors typically rise at approximately five to seven days after exposure to factor, peak at 7 to 21 days, and may persist for years, even in the absence of re-exposure [80,81].

Individuals who develop inhibitors may have an infusion reaction in response to factor administration, although this is uncommon in hemophilia A. More commonly in hemophilia B, individuals who develop inhibitors may have other manifestations such as allergic-type reactions or anaphylaxis to factor infusions, and/or nephrotic syndrome during immune tolerance induction (ITI). (See 'Immune tolerance induction' below.)

SCREENING AND EARLY DIAGNOSISEarly detection of inhibitors is important, both to be aware of the inhibitor when selecting products to treat bleeding, as well as to facilitate early consideration of inhibitor eradication or switching to a different product for prophylaxis. These decisions are discussed separately. (See "Treatment of bleeding and perioperative management in hemophilia A and B", section on 'Inhibitors' and "Hemophilia A and B: Routine management including prophylaxis", section on 'Overview of decision-making'.)

Routine screening and preoperative testing — Experts generally agree that individuals should be screened periodically for inhibitors and tested for inhibitors prior to surgery and/or if factor infusions do not produce the expected increase in factor levels. Surveillance is a best practice approach because in many cases it is the only way to determine whether an inhibitor has developed. Guidance has been provided by the United Kingdom Haemophilia Centre Doctors' Organisation (UKHCDO), which recommends surveillance and testing for inhibitors as follows [40]:

●Hemophilia A – For severe disease, at least every third exposure day or every three months (whichever is sooner) until 20 exposure days have been reached; then every three to six months until 150 exposure days have been reached. For individuals receiving prophylaxis, it may be more practical to measure the trough factor level every three to six months from exposure day 20 until 150 exposure days and only test for an inhibitor if the trough factor level is lower than expected. After exposure day 150, inhibitor testing should continue one to two times per year indefinitely.

For moderate or mild disease, test annually if exposed to factor as well as after any intensive factor exposure (eg, at least five exposure days) or after any surgery. More intensive surveillance may be appropriate if there is a factor VIII mutation with an especially high inhibitor prevalence. (See 'Patient characteristics' above.)

●Hemophilia B – For severe disease, at least every third exposure day or every three months (whichever is sooner) until 20 exposure days have been reached; then every three to six months until 150 exposure days have been reached. After exposure day 150, inhibitor testing is not needed unless clinically indicated; no factor IX inhibitors have been reported beyond exposure day 150. Some experts may change the cutoff to exposure day 50, as few factor IX inhibitors emerge beyond 50 exposure days.

A 2015 United States guideline from the International Immune Tolerance Induction Study Investigators and 2015 guidance from the National Hemophilia Foundation (NHF) Medical and Scientific Advisory Council (MASAC) concur with this approach to inhibitor screening [82,83].

These guidelines also note that additional inhibitor testing is appropriate in individuals with hemophilia A or B in selected circumstances such as the following:

●Before elective procedures that may require factor administration

●If the clinical or laboratory response to factor infusion is suboptimal

●If the patient has an infusion reaction or allergic reaction to factor administration

●If the patient has an increase in bleeding manifestations (increased severity or frequency of bleeding)

While not addressed by guidelines, it is also important to monitor inhibitor titers in individuals with hemophilia A with inhibitors who are receiving emicizumab prophylaxis, especially before elective surgery. This is because inhibitor titers in these individuals may decline to undetectable levels, in which case there is a window during which factor infusions can be used to treat or prevent bleeding. Typically, there is an approximately five- to seven-day window before the anamnestic immune response occurs and the inhibitor titer rises. (See "Hemophilia A and B: Routine management including prophylaxis", section on 'Emicizumab for hemophilia A'.)

A 2015 expert report from the Centers for Disease Control (CDC) in the United States stated that inhibitors are generally under-reported [84]. In an analysis of 12,851 people with hemophilia in the Universal Data Collection surveillance system, only 39 percent of the data forms indicated that inhibitor testing had been performed (average of 46 percent for those with severe hemophilia).

Inhibitor diagnosis and characterization (titer) — Inhibitors may be detected on routine screening, preoperative testing, or testing for a clinical indication such as inadequate response or allergic reaction to factor infusion, as discussed above. (See 'Routine screening and preoperative testing' above.)

Ideally, inhibitor assays are done after a washout of infused factor, when the individual is at their baseline factor level (eg, at least 48 hours after the last dose of factor VIII or 72 hours after the last dose of factor IX for standard half-life products; longer for extended half-life products) or at the trough before the next factor dose [1,40]. This is because infused factor may mask or quench a low titer inhibitor.

The diagnosis of an inhibitor is made using a Bethesda assay, which both identifies the inhibitor and quantifies it. The assay principle uses serial dilutions of patient plasma incubated at 37ºC with pooled normal plasma and measures factor activity using an activated partial thromboplastin time (aPTT)-based assay [85]. Typically, the Nijmegen modification is used (ie, the Nijmegen-Bethesda assay [NBA]); the NBA is considered the gold standard for inhibitor testing [1]. The Nijmegen modification further standardizes the pH and protein concentrations in the assay, which improves specificity and reliability, especially at low inhibitor titers [86,87].

Importantly, this type of aPTT-based assay cannot be used to diagnose or quantify inhibitors in individuals receiving emicizumab. Emicizumab will normalize the aPTT despite the presence of an inhibitor, making it appear that the inhibitor is absent or of markedly lower titer than it actually is. For individuals receiving emicizumab, inhibitor titer must be measured with a chromogenic assay specific for factor VIIIa activity, similar to testing factor VIII levels on emicizumab. (See "Hemophilia A and B: Routine management including prophylaxis", section on 'Emicizumab for hemophilia A' and "Treatment of bleeding and perioperative management in hemophilia A and B".)

The titer of the inhibitor, expressed as Bethesda units (BU), is the reciprocal dilution of patient plasma that results in residual 50 percent factor activity. Thus, an individual with a titer of 20 BU requires 20 serial plasma dilutions to dilute out the inhibitor sufficiently to result in 50 percent activity from the normal plasma. An individual with a titer of 2 BU requires only two serial dilutions (ie, their inhibitor is lower).

●A titer of ≥5 BU is considered a high titer inhibitor. Inhibitors that have demonstrated a high titer are considered high-responding inhibitors, even if the titer subsequently declines below 5 BU.

●A titer <5 BU despite repeated exposures to factor is considered a low-responding inhibitor.

An elevated inhibitor titer (typically defined as a level ≥0.6 BU per mL) should be confirmed promptly with a second sample, especially if the first sample was drawn through a heparinized line [1].

Sources of test interference in Bethesda assays include other antibodies such as a lupus anticoagulant (LA; an antibody that reacts with phospholipids in the clot-based assay and prolongs the aPTT). If testing reveals an LA, pharmacokinetic studies and serial testing over time may help determine whether a specific factor VIII inhibitor is also present [88]. Close consultation with the coagulation laboratory is important to ensure that appropriate testing is performed. (See "Diagnosis of antiphospholipid syndrome", section on 'Antiphospholipid antibody testing'.)

Alternative approaches to inhibitor testing and quantification that use immunoassays to detect antibodies to factor are under development but are not in clinical use [89].

Communication of inhibitor status — It is crucial for clinicians caring for people with hemophilia to have current access to the latest information about their inhibitor status. If an individual with an inhibitor has serious bleeding (spontaneous or traumatic) or requires surgery, the details of their inhibitor status will determine pathways for management. Provision of a wallet card (available through a hemophilia treatment center) with accurate information and/or contact information for the individual's primary hemophilia caregiver facilitates provision of this information to people who do not have immediate access to the latest medical record information for the person with hemophilia.

In the United States, development of a new inhibitor should be reported to the US Food and Drug Administration MedWatch adverse event reporting system.

Genes affected in Hemophilia

• Changes in the F8 gene are responsible for hemophilia A, while mutations in the F9 gene cause hemophilia B. The F8 gene provides instructions for making a protein called coagulation factor VIII. A related protein, coagulation factor IX, is produced from the F9 gene. Coagulation factors are proteins that work together in the blood clotting process. After an injury, blood clots protect the body by sealing off damaged blood vessels and preventing excessive blood loss.

• Mutations in the F8 or F9 gene lead to the production of an abnormal version of coagulation factor VIII or coagulation factor IX, or reduce the amount of one of these proteins. The altered or missing protein cannot participate effectively in the blood clotting process. As a result, blood clots cannot form properly in response to injury. These problems with blood clotting lead to continuous bleeding that can be difficult to control. The mutations that cause severe hemophilia almost completely eliminate the activity of coagulation factor VIII or coagulation factor IX. The mutations responsible for mild and moderate hemophilia reduce but do not eliminate the activity of one of these proteins.

• Another form of the disorder, known as acquired hemophilia, is not caused by inherited gene mutation. This rare condition is characterized by abnormal bleeding into the skin, muscles, or other soft tissues, usually beginning in adulthood. Acquired hemophilia results when the body makes specialized proteins called auto antibodies that attack and disable coagulation factor VIII. The production of auto antibodies is sometimes associated with pregnancy, immune system disorders, cancer, or allergic reactions to certain drugs. In about half of cases, the cause of acquired hemophilia is unknown.