464509

2012
TAH412040620712464509Therapeutic Advances in HematologyC Witmer and G Young

Therapeutic Advances in Hematology Review

Factor VIII inhibitors in hemophilia A: Ther Adv Hematol

(2013) 4(1) 59­–72

rationale and latest evidence DOI: 10.1177/

2040620712464509

© The Author(s), 2012.

Reprints and permissions:
Char Witmer and Guy Young http://www.sagepub.co.uk/
journalsPermissions.nav

Abstract:  Factor VIII (FVIII) replacement therapy is the foundation of treatment in hemophilia
A and is effective unless a patient develops an alloantibody (inhibitor) against exogenous FVIII.
Inhibitor development is currently the most significant treatment complication seen in patients
with hemophilia and is associated with considerable morbidity and a decreased quality of
life. The development of an inhibitor is the result of a complex interaction between a patient’s
immune system and genetic and environmental risk factors. The mainstay of treatment is the
eradication of the inhibitor through immune tolerance. This review summarizes the current
evidence regarding inhibitor risk factors, eradication, and hemostatic bypassing agents.

Keywords:  FVIII, FVIII alloantibodies, hemophilia A, immune tolerance, inhibitors, risk factors

Introduction
Hemophilia A is the most common congenital
severe bleeding disorder and is the result of a defi-
ciency in the clotting protein factor VIII. Factor
VIII (FVIII) deficiency is an X-linked recessive
disorder occurring in 1 in every 5000 male births
without an ethnic predominance [Soucie et al.
1998]. Currently the mainstay of treatment is the
replacement of FVIII with the use of either plasma
or recombinant FVIII concentrates to achieve
hemostasis. FVIII replacement is effective unless
a patient develops an alloantibody (inhibitor)
against the exogenous FVIII. FVIII inhibitors
interfere with the infused factor concentrates ren-
dering them ineffective and necessitating the use
of more costly and less effective alternative hemo-
static agents [Di Minno et al. 2010; Gringeri et al.
2003]. Inhibitor development is currently the
most significant treatment complication seen in
patients with hemophilia. While improvements in
hemostatic agents for patients with inhibitors have
resulted in decreased mortality, inhibitors are still
associated with significant morbidity, including a
higher rate of bleeding complications, increased
disability and a decreased quality of life[Brown
et al. 2009; Darby et al. 2004; Di Minno et al. 2010;
Gringeri et al. 2003; Morfini et al. 2007].
Definition of an inhibitor
An inhibitor is a polyclonal high-affinity immuno-
globulin G (IgG) that is directed against the FVIII
protein [Fulcher et al. 1987; Gilles et al. 1993]. Correspondence to:
Char Witmer, MD, MSCE
IgG4 antibodies are predominant and do not Division of Hematology,
fix complement [Fulcher et al. 1987; Gilles et al. The Children’s Hospital of
Philadelphia, 3501 Civic
1993; Lollar, 2004]. The formation of a FVIII Center Boulevard, CTRB
inhibitor is a T-cell dependent event that includes 11th Floor, Room 11026,
Philadelphia, PA 19104,
antigen-presenting cells, B- and T-helper lym- USA
phocytes [Astermark, 2006]. Antibodies can be witmer@email.chop.edu
Guy Young, MD
either inhibitory or noninhibitory. FVIII contains Division of Hematology/
three A domains (A1, A2, A3), one B domain and Oncology, Children’s
Hospital Los Angeles and
two C domains (C1, C2). Inhibitory antibodies University of Southern
are primarily directed against the A2, A3 and C2 California Keck School of
Medicine, Los Angeles,
domains [Fulcher et al. 1985; Scandella et al. CA, USA
1989]. Antibody binding at these domains results
in steric hindrance blocking functional epitopes
of FVIII [Saint-Remy et al. 2004]. These func-
tional epitopes include FIX, phospholipid and
von Willebrand factor interaction sites. Antibodies
in inhibitor patients can simultaneously target
multiple FVIII epitopes and these epitope targets
can change over time [Fulcher et al. 1988].
FVIII inhibitors are classified based on the kinet-
ics and extent of inhibition of FVIII. Type I inhib-
itors follow second-order kinetics (dose-dependent
linear inhibition) and completely inactivate FVIII.
Type II inhibitors have complex kinetics and
incompletely inactivate FVIII. Type I inhibi-
tors are more common in severe hemophilia.
Type II inhibitors are more common in inhibi-
tor patients with mild hemophilia or in patients
without hemophilia who develop an acquired
FVIII inhibitor.

http://tah.sagepub.com 59
Therapeutic Advances in Hematology 4 (1)
Laboratory characterization of an inhibitor
The most common methods used to detect and
quantify FVIII inhibitors include the Bethesda
assay or the Nijmegen-modified Bethesda assay
[Kasper et al. 1975; Verbruggen et al. 1995].
The International Society on Thrombosis and
Hemostasis FVIII/FIX subcommittee recom-
mend that the Nijmegen-modified Bethesda
assay be used secondary to improved sensitivity
and specificity [Giles et al. 1998]. These assays
only detect inhibitors that reduce FVIII activity
(inhibitory). Both assays utilize serial dilutions
of a patient’s plasma that is incubated with equal
volumes of normal plasma for 2 h at 37°C [Lee
et al. 2005]. The residual factor VIII level of the
incubation mixtures is measured. A positive
result is when there is a significant decrease in
the residual FVIII. The dilutions and residual
factor VIII are plotted against each other and the
inhibitor titer is obtained by linear regression
[Lee et al. 2005]. By definition one Nijmegen-
Bethesda unit reduces the FVIII activity level
by 50%.
There are limitations to laboratory measures of
inhibitors, including a limited sensitivity for low
titer inhibitors [<0.4 Nijmegen-Bethesda unit
(BU)/ml]. There is also a high inter-laboratory
variation in the quantitation of inhibitors with a
high number of false-positive and false-negative
results [Meijer and Verbruggen, 2009]. This is
likely secondary to a variation in how laboratories
carry out their assays (use a mix of the Bethesda
and Nijmegen methods) and a lack of a refer-
ence antibody standard [Meijer and Verbruggen,
2009]. These assays are also better at detecting
and measuring type I inhibitors than type II
inhibitors. Enzyme-linked immunosorbent or flu-
orescent based immune assays can detect both
inhibitory and noninhibitory antibodies and may
have improved detection for low-titer inhibitors
but further validation is needed to support wide-
spread use [Dazzi et al. 1996; Ling et al. 2003;
Zakarija et al. 2011].
Inhibitors are classified into low- or high-responding
inhibitors based on a patient’s peak inhibitor titer
after repeated FVIII exposure. The International
Society on Thrombosis and Hemostasis Scientific
and Standardization committee has recommended
that an inhibitor titer of 5 BU differentiates low-
from high-responding inhibitors [White et al. 2001].
An antibody titer that is persistently below 5 BU
despite repeat challenges with factor VIII is consid-
ered a low-responding inhibitor. A high-responsive
60 http://tah.sagepub.com
inhibitor is applied if the assay has been greater
than 5 BU at any time [White et al. 2001].
Epidemiology
A systematic review regarding the epidemiology
of inhibitors in hemophilia A reported an overall
inhibitor prevalence of 5–7% and when limited to
patients with severe disease the prevalence is
much higher at 12–13% [Wight and Paisley,
2003]. The incidence of new FVIII inhibitors in
patients with severe FVIII deficiency is approxi-
mately 30% [Lusher et al. 1993]. About 60% of
these inhibitors are high titer (>5 BU) and the
remaining are low titer (<5 BU) [Lusher et al.
1993]. Inhibitors are less common in patients
with mild or moderate hemophilia occurring in
approximately 3–13% of patients [Addiego et al.
1993; Ehrenforth et al. 1992; Sultan, 1992]. Most
patients develop an inhibitor within a relatively
short time period of exposure with a median of
9–12 exposure days [Addiego et al. 1993; Bray
et al. 1994; Ehrenforth et al. 1992; Lusher et al.
1993]. A recent study from the UK revealed a
previously unrecognized bimodal peak of inhibi-
tor risk in early childhood and old age [Hay et al.
2011]. Further studies are needed to investigate
this late peak of inhibitor formation and the
mechanism that underlie this.
Clinical presentation
Factor VIII inhibitors can be detected either with
routine laboratory testing or by clinical presenta-
tion. An inhibitor is clinically suspected when a
patient experiences bleeding that does not ade-
quately respond to hemostatic therapy. It is also
prudent to check an inhibitor titer prior to an
invasive procedure to ensure adequate hemosta-
sis. Currently there is no consensus regarding the
routine surveillance for inhibitors. Many provid-
ers check inhibitor titers at regular intervals
within the highest risk period of the first 50 expo-
sure days and then annually [Kempton and
White, 2009]. There can be drawbacks to routine
surveillance, including the detection of transient
inhibitors which can result in significant patient
anxiety.
Genetic risk factors for inhibitor formation
Genetic mutation
A patient’s specific FVIII genetic mutation is the
most significant risk factor for inhibitor formation.

While the overall incidence of inhibitor formation
in patients with severe hemophilia A is approxi-
mately 30%, this proportion varies significantly
among the type of factor VIII mutations. Those
mutations that are null (large deletions, nonsense
mutations and intron 22 inversions) and do not
produce a FVIII protein are associated with the
overall highest rates of inhibitor formation (21–88%)
[Oldenburg et al. 2004]. The intron 22 inversion,
the most common severe FVIII mutation, has an
inhibitor incidence of 21% [Oldenburg et al. 2004].
Large deletions that involve multiple domains
have the highest proportion of inhibitor forma-
tion of 88% [Oldenburg et al. 2004]. Mutations
that result in a loss of FVIII function but retain
some FVIII production (missense and splice
mutations) have a lower risk of inhibitor forma-
tion (3–10%) [Oldenburg et al. 2004]. Of special
interest are small deletions or insertions within
stretches of adenine repeats (poly-A run) that
result in a very low risk of inhibitor formation
(3%) [Oldenburg et al. 2004]. It was discovered
that there is endogenous restoration of the read-
ing frame that results in a small amount of FVIII
production that is likely sufficient to induce tol-
erance [Young et al. 1997].
A recent meta-analysis of FVIII genetic mutation
and inhibitor development included data from 30
different studies with a total of 5383 patients with
severe hemophilia A [Gouw et al. 2012]. To date,
this is the largest study on FVIII genetic mutation
and inhibitor formation providing more precise
estimates of inhibitor risk. Compared with the
risk of inhibitor formation in inversion 22,
large deletions and nonsense mutations had an
increased inhibitor risk with an associated odds
ratio (OR) of 3.6 [95% confidence interval (CI)
2.3–5.7]. The risk of inhibitor formation from
intron 1 inversion (OR 0.9; 95% CI 0.6–1.5) and
splice site mutations (OR 1; 95% CI 0.6–1.5) was
equal to intron 22 inversion. For small deletions/
insertions and missense mutations the risk was
lower (OR 0.5; 95% CI 0.4–0.6).
Race/ethnicity
Patients of African or Hispanic heritage have an
increased risk of inhibitor formation [Aledort
and Dimichele, 1998; Astermark et al. 2001;
Carpenter et al. 2012; Ragni et al. 2009a; Scharrer
et al. 1999]. Early studies demonstrating these
associations were criticized secondary to a lack
of control for FVIII genotype. A recent study
by Miller and colleagues when controlling for
http://tah.sagepub.com 61
C Witmer and G Young
genotype still demonstrated an increased risk of
inhibitor formation in patients of African or
Hispanic descent [Miller et al. 2012]. The mecha-
nisms that account for these racial/ethnic differ-
ences remain unclear. Viel and colleagues found
that a difference in FVIII haplotype between
patients of African heritage and recombinant
FVIII products accounted for this difference [Viel
et al. 2009]. Critics of the study by Viel and col-
leagues report concerns regarding adequate control
of confounding and how the mutational analysis
was completed [Eckhardt et al. 2009a; Lacroix-
Desmazes et al. 2009; Peyvandi et al. 2009; Santos
et al. 2009; Yang et al. 2009]. Further studies are
required to confirm this finding.
Immunologic factors
Individual immune response traits may also affect
a patient’s reaction to exogenous factor VIII.
Explored immunologic factors include the major
histocompatibility complex (MHC) class II sys-
tem and polymorphisms of cytokines [interleukins
(ILs), tumor necrosis factor (TNF)-α]. To date
MHC phenotype has not consistently been dem-
onstrated as a risk factor for inhibitor formation
[Astermark et al. 2006a; Hay et al. 1997;
Oldenburg et al. 1997; Pavlova et al. 2009]. The
Malmo International Brother Study demonstrated
that polymorphisms of the TNF-α gene and IL-10
are associated with an increased risk of inhibitor
formation [Astermark et al. 2006a, 2006b].
Additional studies regarding IL-10 polymor-
phisms have revealed inconsistent results [Bafunno
et al. 2010; Lozier et al. 2011; Pavlova et al. 2009].
Variation in study results is likely secondary to
study design and the specific hemophilia popu-
lation that is being studied. Further studies are
needed to confirm these findings.
Treatment-related risk factors for
inhibitor formation
Inhibitor formation is a complex immune response
that depends not only on the genetic factors dis-
cussed previously but also on treatment-related
risk factors. Treatment-related risk factors that
have been postulated include age at first expo-
sure, the intensity of the first exposure, ‘danger
signals’, prophylaxis, and the type of FVIII product
(recombinant versus plasma derived). Interpretation
of the current studies regarding environmental
risk factors is complicated secondary to retro-
spective designs, variable methodologies and lack
of control for confounding factors.
Therapeutic Advances in Hematology 4 (1)
There are conflicting data regarding age at first
treatment as a risk factor for inhibitor formation.
Two small cohort studies found an inverse corre-
lation between the age (<6 months) of first expo-
sure and inhibitor formation but they were not
controlled for risk factors for inhibitor formation
[Lorenzo et al. 2001; van der Bom et al. 2003].
Additional studies that controlled for known con-
founders (i.e. FVIII genetic mutation, treatment
intensity) were unable to demonstrate an associa-
tion with age [Chalmers et al. 2007; Gouw et al.
2007b; Maclean et al. 2011; Santagostino et al.
2005]. It is likely that the association between
inhibitor development and age of first factor
exposure is explained by early intensive therapy
[Kempton and White, 2009].
The intensity of the first FVIII exposure is postu-
lated to be a risk factor for inhibitor formation
because significant cell injury or inflammation
leads to immunologic ‘danger signals’ that stimu-
late antigen-presenting cells and amplify an immu-
nologic response which could promote inhibitor
development [Gallucci and Matzinger, 2001;
Kono and Rock, 2008; Matzinger, 2002]. Several
studies have demonstrated an increased rate of
inhibitor formation after intensive treatment (i.e.
surgical procedure or high-frequency treatment)
[Gouw et al. 2007a, 2007b; Maclean et al. 2011;
Ragni et al. 2009a]. However, a case–control study
by Santagostino and colleagues did not confirm
these findings [Santagostino et al. 2005]. Overall it
does appear that there is an increased risk of inhib-
itor formation after an intense initial FVIII expo-
sure. This association is not a reason to withhold
life-saving therapy but should prompt a clinician at
the time of a significant early exposure to monitor
closely for the development of an inhibitor.
There is an ongoing debate regarding the immu-
nogenicity of factor products with a concern that
recombinant products have an increased risk of
inhibitor formation over that of plasma-derived
products. This increased immunogenicity is
hypothesized to be secondary to alterations in
posttranslational modifications of FVIII and a lack
of von Willebrand binding [Dasgupta et al. 2007;
Grancha et al. 2011; Hironaka et al. 1992; Qadura
et al. 2009]. There are conflicting reports in the lit-
erature that have led to this state of uncertainty. In
comparing studies in previously untreated patients
(PUPs) there is a difference in the overall reported
cumulative incidence of inhibitor formation in
plasma derived (10.3%) versus recombinant
(28.7%) [Mannucci et al. 2012]. The difficulty in
62 http://tah.sagepub.com
comparing the cumulative incidence from these
studies is the high degree of heterogeneity in patient
inhibitor risk factors, study methodology, fre-
quency of inhibitor testing and length of follow up
[Mannucci et al. 2012]. Two recent systematic
reviews attempted to control for this known hetero-
geneity, and were unable to demonstrate a differ-
ence in inhibitor formation [Franchini et al. 2012;
Iorio et al. 2010]. In the review by Iorio and col-
leagues, study design, study period, testing fre-
quency and median follow up explained most of the
variability and the source of factor concentrate lost
statistical significance [Iorio et al. 2010]. To address
this very important clinical question a prospective
international randomized clinical trial (SIPPET –
Survey of Inhibitors in Plasma Product Exposed
Toddlers) is currently enrolling patients and is com-
paring inhibitor incidence in previously untreated
patients exposed to either plasma or recombinant
factor products [Mannucci et al. 2007].
The use of early prophylaxis at a young age has
been proposed as a mechanism to induce toler-
ance through early controlled antigen exposure
and to minimize the possibility that the first FVIII
exposure is in the setting of a ‘danger signal’. Two
cohort studies, an Italian cohort and the CANAL
(concerted action on neutralizing antibodies in
severe hemophilia A) study, reported a decreased
risk of inhibitor formation for patients receiving
prophylaxis versus on-demand therapy [Gouw
et al. 2007b; Santagostino et al. 2005]. This was
not confirmed in a cohort study from the UK
[Maclean et al. 2011]. A small prospective pilot
study in 26 PUPs from Germany were treated
with 25 IU/kg/week as soon as bleeding tendency
manifested [Kurnik et al. 2010]. The prevalence
of inhibitor formation was compared with histori-
cal controls with a significant decrease in inhibi-
tor formation in the study group with only 1/26
(3%) developing inhibitors versus 14/30 (46%)
with an associated OR of 0.048 (95% CI 0.001–
0.372) [Kurnik et al. 2010].This study, however,
has been criticized for not having contemporane-
ous controls, and the quite spectacular results
need to be confirmed in prospective studies.
Table 1 provides a summary of all the known cur-
rent risk factors for inhibitor formation.
Stratification of inhibitor risk
With the advancement in the understanding of risk
factors that underlie inhibitor formation, efforts
have been made to create a clinical scoring system
 C Witmer and G Young

Table 1.  Risk factors for inhibitor formation in hemophilia A.

Genetic risk factors

Genetic mutation: the highest risk is found for null mutations (i.e. large deletions, nonsense mutations and
the intron 22 inversion)
Family history of an inhibitor
African or Hispanic race/ethnicity
Immune response traits: explored immunologic factors include the major histocompatibility complex class
II system and polymorphisms of cytokines (interleukins, tumor necrosis factor α)
Environmental risk factors
Intensity of the first factor VIII exposure: surgical procedure, high frequency treatment
Inconclusive factors further study is needed: - Source of factor VIII: plasma-derived versus recombinant
factor products
                                        - Early prophylaxis: to prevent inhibitor formation

to quantify an individual’s risk. Ter Avest and col- Eradication of inhibitors

leagues created a clinical scoring system that uti-
lizes three clinical factors (family history of
inhibitor, gene mutation and intensive treatment
at initial exposure) to predict inhibitor formation
[ter Avest et al. 2008]. The risk score is cumula-
tive and includes two points for a positive family
history of inhibitors, two points for a high-risk
gene mutation and three points if the initial FVIII
treatment is intensive. Patients without any risk
factors (risk score=0 points) had an inhibitor inci-
dence of 6%, 23% in those with two points and
57% in those patients with more than three points
[ter Avest et al. 2008]. The limitations of this clin-
ical scoring system include the use of a primarily
white cohort and the lack of inclusion of other
well established risk factors.
While a risk score can help identify patients at the
highest risk for inhibitor formation we are still left
with a quandary as to how to effectively minimize
this risk. As mentioned previously, some propose
the institution of early prophylaxis, although fur-
ther data are needed to confirm these findings.
Others have proposed the use of alternative
hemostatic products to delay FVIII exposure.
Rivard and colleagues completed a prospective
trial in 11 pediatric patients with severe hemo-
philia using recombinant factor VIIa to try and
postpone exposure to FVIII until after 2 years
of age [Rivard et al. 2005]. Owing to inadequate
hemostasis with recombinant factor VIIa, espe-
cially in mouth bleeding, only 3 out of 11 chil-
dren could postpone FVIII exposure until after
2 years of age [Rivard et al. 2005], making this
therapeutic option limited. Challenges remain as
to the most effective manner in which to mini-
mize a patient’s risk of inhibitor formation and
further studies are required.
Owing to the significant clinical ramifications of
inadequate hemostasis in patients with high-titer
FVIII inhibitors, the overarching therapeutic goal is
eradication of the inhibitor. At this time the only
proven method for eradication is immune tolerance
induction (ITI). ITI is the regular infusion of FVIII
to induce FVIII antigen-specific tolerance. There is
currently no consensus regarding the specifics of
ITI treatment, including the factor product source
(plasma derived versus recombinant), factor dose,
timing or the use of immune modulation.
Multiple immune tolerance registries were estab-
lished to help determine patient- and treatment-
related factors associated with immune tolerance
outcome [Coppola et al. 2009; Dimichele, 2009;
Haya et al. 2001; Lenk, 2000; Mariani et al. 1994].
Overall reported success rates in these registries
ranged from 51% to 79% [Coppola et al. 2009;
Dimichele, 2009; Haya et al. 2001; Lenk, 2000;
Mariani et al. 1994]. This wide range in success
rates is likely secondary to a lack of standardiza-
tion in study methodologies, treatment protocols,
and eradication definitions. Consistently across
these registries a patient’s peak historical FVIII
inhibitor titer (<200 BU) and the inhibitor titer at
the time of ITI induction (<10 BU) were associ-
ated with successful immune tolerance. Other pre-
dictors have been identified but have not been
consistent across studies. Table 2 provides a com-
plete list of predictors for ITI.
There are conflicting data regarding the ITI fac-
tor VIII dose and success of ITI. The International
Immune Tolerance Registry demonstrated
improved ITI success with high doses (200 IU/kg)
while the North American and Spanish Immune
Tolerance Registries demonstrated improved

http://tah.sagepub.com 63
Therapeutic Advances in Hematology 4 (1)
Table 2.  Predictors of immune tolerance induction
Predictors of success
Pre-ITI inhibitor titer <10 BU [Coppola et al. 2009;
Dimichele, 2009; Haya et al. 2001; Lenk et al. 2000;
Mariani et al. 1994]
Historical peak inhibitor titer <200 BU [Coppola et al.
2009; Dimichele, 2009; Haya et al. 2001; Lenk et al.
2000; Mariani et al. 1994]
Low factor VIII dose for ITI* [Dimichele, 2009;
Haya et al. 2001]
Factor VIII genotype (low-risk mutations: small
insertions, deletions or missense) [Coppola et al. 2009]
*There are conflicting results regarding a low factor VIII dose for immune tolerance induction and whether it is a predictor
of failure or success.
BU, Bethesda unit; ITI, immune tolerance induction.
success with lower dosing strategies [Dimichele,
2009; Haya et al. 2001; Mariani et al. 1994]. A
meta-analysis of the International and North
American Registries found that FVIII dosing
did not influence ITI success [Kroner, 1999].
To address this discrepancy the International
Immune Tolerance Study was completed [Hay
and DiMichele, 2012]. This multicenter rand-
omized clinical trial compared high-dose
(200 IU/kg/day) with low-dose (50 IU/kg three
times/week) regimens in patients with severe
hemophilia A and high-titer inhibitors (>5 BU).
Product choice was left up to the managing clini-
cian; 90% received recombinant FVIII prod-
ucts. This study was stopped early secondary to
increased bleeding events in the low-dose arm.
There was no difference in the proportion of
ITI success between the two arms but the time
to achieve ITI success was shorter in the high-
dose arm. Because the study was stopped early
it lacked statistical power to demonstrate thera-
peutic equivalence below the 30% boundary of
equivalence. It appears that a high-dose strategy
achieves tolerance at a faster rate and this may
explain the associated decreased bleeding rate.
Controversy also remains regarding the source of
product for immune tolerance and the impact on
ITI success. The role of von Willebrand factor in
achieving tolerance is unclear. The current pub-
lished data are limited by small sample size, retro-
spective design and lack of control for known
confounders. A recent comprehensive literature
review in comparing these studies found no differ-
ence in ITI success based on factor product, with
success rates of 75–87% in plasma-derived FVIII,
68–91% in von Willebrand factor containing
64 http://tah.sagepub.com
Predictors of failure
Interval >5 years between diagnosis of inhibitor
and start of ITI [Kroner, 1999]
ITI interruption [Lenk et al. 2000]
Low factor VIII dose for ITI* [Mariani et al. 1994]
High peak titer on ITI [Dimichele, 2009; Haya et al.
2001; Lenk et al. 2000; Mariani et al. 1994]
products and 63–82% in recombinant products
[Franchini and Lippi, 2011]. The RESIST (Rescue
Immunotolerance Study naive) trial is currently
enrolling patients and is a randomized clinical trial
that compares ITI success in high-risk patients
(age >6 years, peak historical inhibitor >200, pre-
ITI titer >10 BU, time between inhibitor forma-
tion and ITI >2 years) using von Willebrand factor
containing FVIII versus recombinant FVIII
[Gringeri, 2007]. At this time the available evi-
dence does not support the superiority of one
product source over another for use in ITI.
Although not common to most ITI protocols the
use of immune modulation has been reported as a
method to improve tolerance success. In Sweden
the Malmo protocol uses a combination of immu-
noabsorption (to acutely decreased the FVIII
inhibitor titer), cyclophosphamide, intravenous
immunoglobulin and daily high-dose FVIII
[Nilsson et al. 1988]. The reported benefit appears
to be decreased time to tolerance but the overall
success rate is comparable to ITI protocols with-
out the risks associated with immune modulation
[Berntorp et al. 2000]. Recently there has been an
interest in the use of the monoclonal anti-CD20
antigen (rituximab) that inhibits B cells and inter-
feres with IgG production. A phase II single-arm
clinical trial used rituximab as a single agent in
patients with high-titer inhibitors whose condi-
tion had failed to respond to prior ITI attempts.
The results of this trial were presented as an oral
presentation at the 2011 American Society of
Hematology Annual Meeting [Leissinger et al.
2011b]. The authors reported that only 3 out of
16 subjects enrolled (18.8%) had a major response
(fall in the inhibitor to <5 BU without an increase

in the inhibitor titer after rechallenge to FVIII)
[Leissinger et al. 2011b]. It appears that as a solo
agent in patients previously treated with inhibi-
tors, rituximab had a small effect but further stud-
ies are needed to determine if rituximab could
be more effective if used with ITI.
New strategies for immunomodulation
in FVIII inhibitors
Currently there is ongoing research in mouse
models focusing on novel products and methods
to modulate the immune response to factor VIII
[Miao, 2010; Waters and Lillicrap, 2009]. Multiple
methods have been used to manipulate antigen
presentation, including oral or nasal administra-
tion of FVIII peptides, infusion of immature den-
dritic cells or apoptotic fibroblast cells [Qadura
et al. 2008; Ragni et al. 2009b; Rawle et al. 2006;
Su et al. 2010]. While these methods demonstrated
a decrease in the immune response to FVIII they
were unable to completely mitigate this response.
Diverse strategies have also been explored that
evade the immune response to FVIII. These include
the preparation of less immunogenic FVIII pro-
teins or through the ongoing exposure to FVIII
through novel gene therapy methods including
neonatal gene transfer, ex vivo transduction of
hematopoietic stem cells or site-specific
FVIII gene expression in platelets [Miao, 2010].
Additional focus has also been placed on targeted
immunosuppression of either the T- or B-cell
pathways. The most successful of these immuno-
suppressive efforts include those that increase
T-regulatory cells using either protein replace-
ment or gene therapy [Miao, 2010]. While much
of this research is currently in mouse models, it is
anticipated that many of these protocols warrant
further consideration for clinical translational
research. At this time it is unclear whether any of
these strategies could be used alone, in combina-
tion or in concert with current ITI management.
The ultimate goal for immunomodulation ther-
apy is a therapy that allows a patient’s immune
system to function appropriately while mediating
long-term FVIII tolerance.
Hemostatic agents for patients with an
inhibitor
Treatment of bleeding in a patient with an inhibi-
tor is based on the classification of the inhibitor. A
low-titer and low-responding inhibitor (<5 BU)
can commonly be overcome with higher doses
of FVIII, while patients with high-titer/
http://tah.sagepub.com 65
C Witmer and G Young
high-responding inhibitors must be treated with
bypassing agents. In general, bypassing agents
can achieve hemostasis but they are not as effec-
tive as FVIII replacement in patients without an
inhibitor. Options for bypass therapy include an
activated prothrombin complex concentrate
(aPCC; FEIBA, FVIII inhibitor bypassing activ-
ity; Baxter AG, Vienna, Austria) or recombinant
factor VIIa (rFVIIa; NovoSeven, Novo Nordisk
A/S, Bagsvaerd, Denmark). Porcine factor VIII is
not currently available although a recombinant
version is in development [Kempton et al. 2012].
Both rFVIIa and aPCCs have been demonstrated
to be effective in the treatment of bleeding for
patients with inhibitors [Buchanan and Kevy,
1978; Hilgartner and Knatterud, 1983; Kurczynski
and Penner, 1974; Lusher et al. 1998; Shapiro
et al. 1998; Sjamsoedin et al. 1981]. A randomized
crossover trial of rFVIIa versus FEIBA assessed
patient reported efficacy 6 h after treatment and
did not demonstrate statistical equivalence
between the two products. [Astermark et al. 2007].
However, the confidence interval of the difference
only slightly exceeded the 15% boundary (–11.4%
to 15.7%), p = 0.59[Astermark et al. 2007]. The
efficacy between products was rated quite dif-
ferently by a substantial portion of subjects
[Astermark et al. 2007.
In determining which bypassing product to use in
an individual patient it is important to consider
multiple factors, including the need to avoid
FVIII exposure to prevent anamnesis and a
patient’s individual response to therapy. In a
patient with a newly diagnosed inhibitor whom
one is waiting for the FVIII inhibitor titer to fall
before initiating ITI it is prudent to use rFVIIa
because aPCCs contain a small amount of FVIII
and have been shown to increase the inhibitor
titer. It is well recognized that there can be signifi-
cant patient variability in their individual hemo-
static response to bypassing agents. In some
patients bleeding can be unresponsive to mono-
therapy and may require alternating products
[Gringeri et al. 2011a; Schneiderman et al. 2004].
Secondary to the risk of developing thrombosis
or disseminated intravascular coagulation from
alternating bypassing agents, this therapy should
be used with caution and only in an inpatient
setting [Ingerslev and Sorensen, 2011].
It is clear that prophylaxis in patients with severe
hemophilia (without an inhibitor) is effective in
preventing bleeding [Gringeri et al. 2011b; Manco-
Johnson et al. 2007]. Recently two trials have
Therapeutic Advances in Hematology 4 (1)
demonstrated the efficacy of bypassing agents as
secondary prophylaxis for patients with inhibitors
[Konkle et al. 2007; Leissinger et al. 2011a]. The
first trial compared the efficacy of daily rFVIIa
prophylaxis (90 μg/kg versus 270 μg/kg) in 22
inhibitor patients and demonstrated decreased
bleeding at both dose levels (45% versus 59%)
[Konkle et al. 2007]. Subsequent analysis also
demonstrated an improvement in quality of life
[Hoots et al. 2008]. More recently, a randomized
crossover clinical trial compared the use of an
aPCC (85 units/kg on three nonconsecutive days
a week) with on-demand therapy and demon-
strated a 62% overall reduction in all bleeding
episodes [Leissinger et al. 2011a]. It is unclear
which inhibitor patients should be considered for
prophylaxis and this should be determined on a
case by case basis. It is recommended that proph-
ylaxis be considered for patients whose condition
has failed to respond to ITI and who have recur-
rent significant bleeding (i.e. a target joint or life-
threatening hemorrhages) [Young et al. 2011].
The use of bypassing agents for prophylaxis while
a patient is undergoing an initial course of ITI
should be done with caution because a patient’s
FVIII level could improve while receiving a
bypassing agent which could be prothrombotic
[Young et al. 2011]. This is the subject of an ongo-
ing clinical trial.
Inhibitors in mild hemophilia A
As discussed previously inhibitor development in
mild hemophilia is less common then severe
hemophilia with an estimated incidence of
3–13% [Addiego et al. 1993; Ehrenforth et al.
1992; Sultan, 1992]. Patients with mild hemo-
philia form inhibitors against the exogenous
infused factor VIII and commonly there is inhibi-
tor cross reactivity against the patient’s endoge-
nous FVIII decreasing a patient’s baseline FVIII
level. This decrease changes the patient’s bleed-
ing phenotype from mild to moderate or severe.
Inhibitors can be detected through routine
screening or with a change in a patient’s bleeding
pattern. Some patients may have bleeding mani-
festations that are more commonly seen in
patients with an acquired inhibitor, including
extensive mucocutaneous or urogenital bleed-
ing. Others may experience bleeding more com-
mon to hemophilia with joint and intramuscular
hemorrhage. Both type I and II inhibitors have
been reported in mild hemophilia but there
appears to be a predominance of type II inhibi-
tors [Franchini et al. 2010].
66 http://tah.sagepub.com
Risk factors for inhibitor formation in mild hemo-
philia include later age with first factor VIII
exposure, intensity of the factor exposure, family
history of inhibitors and type of genetic mutation
[Eckhardt et al. 2009b; Hay et al. 1998; Kempton
et al. 2010; Mauser-Bunschoten et al. 2012;
Sharathkumar et al. 2003]. Multiple studies have
reported an association between the Arg593Cys
genetic mutation and an increased risk of inhibitor
formation [Eckhardt et al. 2009b; Hay et al. 1998;
Mauser-Bunschoten et al. 2012]. However, in a
recent study from the USA the Arg593Cys genetic
mutation was not associated with an increased
risk of inhibitor formation [Kempton et al. 2010].
HLA genotype has also been explored as a risk fac-
tor for inhibitor formation in patients with mild
hemophilia with the Arg593Cys genetic mutation
and no association was found [Bril et al. 2004].
There is no established standard of care for the
treatment of patients with mild hemophilia and
an inhibitor. Spontaneous resolution has been
reported in up to 60% of cases after a median of
9 months (range 0.5–46) [Hay et al. 1998].
However, 75% of patients with spontaneous reso-
lution experienced anamnesis with repeat FVIII
exposure [Hay et al. 1998]. Eradication has also
been achieved with the use of immune tolerance
by itself or combined with immune modulation
(prednisone, cyclophosphamide or rituximab)
[Franchini et al. 2008; Hay et al. 1998; Robbins
et al. 2001; Vlot et al. 2002]. The primary treatment
strategy for patients with mild hemophilia should
be inhibitor prevention. A desmopressin (DDAVP)
trial should be completed for all patients with mild
hemophilia. DDAVP should be used preferentially
over factor products in those who are responders.
Mutation analysis and family history can also be
used to identify patients at the highest risk.
Conclusion
In conclusion, the development of an inhibitor is
the most significant treatment complication in
hemophilia A and is the result of complex inter-
action between a patient’s immune system and
genetic and environmental risk factors. Great
strides have been made in understanding the
important risk factors for inhibitor development,
including delineation of risk by genetic mutation,
ethnic/racial differences, immunologic factors, and
intensity of early factor exposure, but further elu-
cidation is needed. Questions remain regarding
the product source and the risk of inhibitor for-
mation and we await the results of the SIPPET

trial to help provide further clarification
[Mannucci et al. 2007]. Data regarding early
prophylaxis and inhibitor prevention are intrigu-
ing but prospective studies are needed to confirm
these findings. ITI is considered standard of care
for inhibitor eradication and a recent randomized
clinical trial demonstrated that a high-dose (200
U/kg daily) regimen achieved tolerance faster and
with less bleeding when compared with a low-dose
(50 U/kg three times/week) regimen [Hay and
DiMichele, 2012]. Uncertainty continues about
the ITI product source, timing, dosing regimen,
and the use of immune modulation. There is also
new evidence to support the use of prophylaxis
with a bypassing agent in patients with inhibitors,
although it is unclear which inhibitor patients are
the best candidates for this therapy [Konkle et al.
2007; Leissinger et al. 2011a; Young et al. 2011].
While significant progress has been made in the
care of patients with inhibitors there remain many
unanswered questions. Ideally in the future we
will be able to accurately predict a patient’s indi-
vidual risk of inhibitor formation and accordingly
modify their individual treatment plan to mini-
mize or abolish their risk of inhibitor formation.
Funding
This review article received no specific grant from
any funding agency in the public, commercial or
not-for-profit sectors.
Conflict of interest statement
GY has received honoraria as a speaker for Baxter
and Novo Nordisk. CW has no conflicts of inter-
est to declare.
References
Addiego, J., Kasper, C., Abildgaard, C., Hilgartner, M.,
Lusher, J., Glader, B. et al. (1993) Frequency of
inhibitor development in haemophiliacs treated with
low-purity factor VIII. Lancet 342: 462–464.
Aledort, L. and Dimichele, D. (1998) Inhibitors occur
more frequently in African-American and Latino
haemophiliacs. Haemophilia 4: 68.
Astermark, J. (2006) Why do inhibitors develop?
Principles of and factors influencing the risk for
inhibitor development in haemophilia. Haemophilia
12(Suppl. 3): 52–60.
Astermark, J., Berntorp, E., White, G. and Kroner, B.
(2001) The Malmo International Brother Study
(MIBS): further support for genetic predisposition
to inhibitor development in hemophilia patients.
Haemophilia 7: 267–272.
http://tah.sagepub.com 67
C Witmer and G Young
Astermark, J., Donfield, S., DiMichele, D.,
Gringeri, A., Gilbert, S., Waters, J. et al. (2007)
A randomized comparison of bypassing agents in
hemophilia complicated by an inhibitor: the FEIBA
NovoSeven Comparative (FENOC) Study. Blood 109:
546–551.
Astermark, J., Oldenburg, J., Carlson, J., Pavlova, A.,
Kavakli, K., Berntorp, E. et al. (2006a)
Polymorphisms in the TNFA gene and the risk of
inhibitor development in patients with hemophilia A.
Blood 108: 3739–3745.
Astermark, J., Oldenburg, J., Pavlova, A., Berntorp, E.
and Lefvert, A. (2006b) Polymorphisms in the
IL10 but not in the IL1beta and IL4 genes are
associated with inhibitor development in patients with
hemophilia A. Blood 107: 3167–3172.
Bafunno, V., Santacroce, R., Chetta, M., D’Andrea, G.,
Pisanelli, D., Sessa, F. et al. (2010) Polymorphisms in
genes involved in autoimmune disease and the risk of
FVIII inhibitor development in Italian patients with
haemophilia A. Haemophilia 16: 469–473.
Berntorp, E., Astermark, J. and Carlborg, E. (2000)
Immune tolerance induction and the treatment of
hemophilia. Malmo protocol update. Haematologica
85(10 Suppl.): 48–50; discussion 50-51.
Bray, G., Gomperts, E., Courter, S., Gruppo, R.,
Gordon, E., Manco-Johnson, M. et al. (1994)
A multicenter study of recombinant factor VIII
(recombinate): safety, efficacy, and inhibitor risk in
previously untreated patients with hemophilia A. The
Recombinate Study Group. Blood 83: 2428–2435.
Bril, W., MacLean, P., Kaijen, P., van den Brink, E.,
Lardy, N., Fijnvandraat, K. et al. (2004) HLA
class II genotype and factor VIII inhibitors in mild
haemophilia A patients with an Arg593 to Cys
mutation. Haemophilia 10: 509–514.
Brown, T., Lee, W., Joshi, A. and Pashos, C. (2009)
Health-related quality of life and productivity impact
in haemophilia patients with inhibitors. Haemophilia
15: 911–917.
Buchanan, G. and Kevy, S. (1978) Use of
prothrombin complex concentrates in hemophiliacs
with inhibitors: clinical and laboratory studies.
Pediatrics 62: 767–774.
Carpenter, S., Michael Soucie, J., Sterner, S. and
Presley, R. (2012) Increased prevalence of inhibitors
in Hispanic patients with severe haemophilia A
enrolled in the Universal Data Collection database.
Haemophilia 18: e260–265.
Chalmers, E., Brown, S., Keeling, D., Liesner, R.,
Richards, M., Stirling, D. et al. (2007) Early factor
VIII exposure and subsequent inhibitor development
in children with severe haemophilia A. Haemophilia
13: 149–155.
Therapeutic Advances in Hematology 4 (1)
Coppola, A., Margaglione, M., Santagostino, E.,
Rocino, A., Grandone, E., Mannucci, P. et al. (2009)
Factor VIII gene (F8) mutations as predictors of
outcome in immune tolerance induction of hemophilia
A patients with high-responding inhibitors. J Thromb
Haemost 7: 1809–1815.
Darby, S., Keeling, D., Spooner, R., Wan Kan, S.,
Giangrande, P., Collins, P. et al. (2004) The
incidence of factor VIII and factor IX inhibitors in the
hemophilia population of the UK and their effect on
subsequent mortality, 1977-99. J Thromb Haemost 2:
1047–1054.
Dasgupta, S., Repesse, Y., Bayry, J., Navarrete, A.,
Wootla, B., Delignat, S. et al. (2007) VWF protects
FVIII from endocytosis by dendritic cells and
subsequent presentation to immune effectors. Blood
109: 610–612.
Dazzi, F., Tison, T., Vianello, F., Radossi, P.,
Zerbinati, P., Carraro, P. et al. (1996) High incidence
of anti-FVIII antibodies against non-coagulant
epitopes in haemophilia A patients: a possible role for
the half-life of transfused FVIII. Br J Haematol 93:
688–693.
Dimichele, D. (2009) The North American Immune
Tolerance Registry: contributions to the thirty-
year experience with immune tolerance therapy.
Haemophilia 15: 320–328.
Di Minno, M., Di Minno, G., Di Capua, M.,
Cerbone, A. and Coppola, A. (2010) Cost of care of
haemophilia with inhibitors. Haemophilia 16: e190–e201.
Eckhardt, C., Kamphuisen, P. and Fijnvandraat, K.
(2009a) Inhibitors of factor VIII in hemophilia.
N Engl J Med 361: 309; author reply 310.
Eckhardt, C., Menke, L., van Ommen, C., van der
Lee, J., Geskus, R., Kamphuisen, P. et al. (2009b)
Intensive peri-operative use of factor VIII and the
Arg593-->Cys mutation are risk factors for inhibitor
development in mild/moderate hemophilia A.
J Thromb Haemost 7: 930–937.
Ehrenforth, S., Kreuz, W., Scharrer, I., Linde, R.,
Funk, M., Gungor, T. et al. (1992) Incidence of
development of factor VIII and factor IX inhibitors in
haemophiliacs. Lancet 339: 594–598.
Franchini, M., Favaloro, E. and Lippi, G. (2010)
Mild hemophilia A. J Thromb Haemost 8: 421–432.
Franchini, M. and Lippi, G. (2011) Immune
tolerance induction for patients with severe
hemophilia A: a critical literature review. J Thromb
Thrombolysis 32: 439–447.
Franchini, M., Mengoli, C., Lippi, G., Targher, G.,
Montagnana, M., Salvagno, G. et al. (2008) Immune
tolerance with rituximab in congenital haemophilia
with inhibitors: a systematic literature review based on
individual patients' analysis. Haemophilia 14: 903–912.
68 http://tah.sagepub.com
Franchini, M., Tagliaferri, A., Mengoli, C. and
Cruciani, M. (2012) Cumulative inhibitor incidence
in previously untreated patients with severe
hemophilia A treated with plasma-derived versus
recombinant factor VIII concentrates: a critical
systematic review. Crit Rev Oncol Hematol 81:
82–93.
Fulcher, C., de Graaf Mahoney, S., Roberts, J.,
Kasper, C. and Zimmerman, T. (1985) Localization
of human factor FVIII inhibitor epitopes to two
polypeptide fragments. Proc Natl Acad Sci U S A 82:
7728–7732.
Fulcher, C., de Graaf Mahoney, S. and Zimmerman, T.
(1987) FVIII inhibitor IgG subclass and
FVIII polypeptide specificity determined by
immunoblotting. Blood 69: 1475–1480.
Fulcher, C., Lechner, K. and de Graaf Mahoney, S.
(1988) Immunoblot analysis shows changes in factor
VIII inhibitor chain specificity in factor VIII inhibitor
patients over time. Blood 72: 1348–1356.
Gallucci, S. and Matzinger, P. (2001) Danger signals:
SOS to the immune system. Curr Opin Immunol 13:
114–119.
Giles, A., Verbruggen, B., Rivard, G., Teitel, J.
and Walker, I. (1998) A detailed comparison of the
performance of the standard versus the Nijmegen
modification of the Bethesda assay in detecting factor
VIII:C inhibitors in the haemophilia A population of
Canada. Association of Hemophilia Centre Directors
of Canada. Factor VIII/IX Subcommittee of Scientific
and Standardization Committee of International
Society on Thrombosis and Haemostasis. Thromb
Haemost 79: 872–875.
Gilles, J., Arnout, J., Vermylen, J. and Saint-Remy, J.
(1993) Anti-factor VIII antibodies of hemophiliac
patients are frequently directed towards nonfunctional
determinants and do not exhibit isotypic restriction.
Blood 82: 2452–2461.
Gouw, S., van den Berg, H., le Cessie, S. and van
der Bom, J. (2007a) Treatment characteristics and
the risk of inhibitor development: a multicenter
cohort study among previously untreated patients
with severe hemophilia A. J Thromb Haemost 5:
1383–1390.
Gouw, S., van den Berg, H., Oldenburg, J.,
Astermark, J., de Groot, P., Margaglione, M. et
al. (2012) F8 gene mutation type and inhibitor
development in patients with severe hemophilia A:
systematic review and meta-analysis. Blood 119:
2922–2934.
Gouw, S., van der Bom, J. and Marijke van den Berg, H.
(2007b) Treatment-related risk factors of inhibitor
development in previously untreated patients with
hemophilia A: the CANAL cohort study. Blood 109:
4648–4654.

Grancha, S., Navajas, R., Maranon, C., Paradela, A.,
Albar, J. and Jorquera, J. (2011) Incomplete tyrosine
1680 sulphation in recombinant FVIII concentrates.
Haemophilia 17: 709–710.
Gringeri, A. (2007) VWF/FVIII concentrates in
high-risk immunotolerance: the RESIST study.
Haemophilia 13(Suppl. 5): 73–77.
Gringeri, A., Fischer, K., Karafoulidou, A.,
Klamroth, R., Lopez-Fernandez, M. and
Mancuso, E. (2011a) Sequential combined bypassing
therapy is safe and effective in the treatment of
unresponsive bleeding in adults and children with
haemophilia and inhibitors. Haemophilia 17: 630–635.
Gringeri, A., Lundin, B., von Mackensen, S.,
Mantovani, L. and Mannucci, P. (2011b) A
randomized clinical trial of prophylaxis in children
with hemophilia A (the ESPRIT Study). J Thromb
Haemost 9: 700–710.
Gringeri, A., Mantovani, L., Scalone, L. and
Mannucci, P. (2003) Cost of care and quality of
life for patients with hemophilia complicated by
inhibitors: the COCIS Study Group. Blood 102:
2358–2363.
Hay, C. and DiMichele, D. (2012) The principal
results of the International Immune Tolerance Study: a
randomized dose comparison. Blood 119: 1335–1344.
Hay, C., Ludlam, C., Colvin, B., Hill, F., Preston, F.,
Wasseem, N. et al. (1998) Factor VIII inhibitors
in mild and moderate-severity haemophilia A. UK
Haemophilia Centre Directors Organisation. Thromb
Haemost 79: 762–766.
Hay, C., Ollier, W., Pepper, L., Cumming, A.,
Keeney, S., Goodeve, A. et al. (1997) HLA class II
profile: a weak determinant of factor VIII inhibitor
development in severe haemophilia A. UKHCDO
Inhibitor Working Party. Thromb Haemost 77:
234–237.
Hay, C., Palmer, B., Chalmers, E., Liesner, R.,
Maclean, R., Rangarajan, S. et al. (2011) Incidence
of factor VIII inhibitors throughout life in severe
hemophilia A in the United Kingdom. Blood 117:
6367–6370.
Haya, S., Lopez, M., Aznar, J. and Batlle, J. (2001)
Immune tolerance treatment in haemophilia patients
with inhibitors: the Spanish Registry. Haemophilia 7:
154–159.
Hilgartner, M. and Knatterud, G. (1983) The
use of factor eight inhibitor by-passing activity
(FEIBA immuno) product for treatment of bleeding
episodes in hemophiliacs with inhibitors. Blood 61:
36–40.
Hironaka, T., Furukawa, K., Esmon, P., Fournel, M.,
Sawada, S., Kato, M. et al. (1992) Comparative study
of the sugar chains of factor VIII purified from human
http://tah.sagepub.com 69
C Witmer and G Young
plasma and from the culture media of recombinant baby
hamster kidney cells. J Biol Chem 267: 8012–8020.
Hoots, W., Ebbesen, L., Konkle, B., Auerswald, G.,
Roberts, H., Weatherall, J. et al. (2008) Secondary
prophylaxis with recombinant activated factor VII
improves health-related quality of life of haemophilia
patients with inhibitors. Haemophilia 14: 466–475.
Ingerslev, J. and Sorensen, B. (2011) Parallel use of
by-passing agents in haemophilia with inhibitors: a
critical review. Br J Haematol 155: 256–262.
Iorio, A., Halimeh, S., Holzhauer, S., Goldenberg, N.,
Marchesini, E., Marcucci, M. et al. (2010) Rate
of inhibitor development in previously untreated
hemophilia A patients treated with plasma-derived or
recombinant factor VIII concentrates: a systematic
review. J Thromb Haemost 8: 1256–1265.
Kasper, C., Aledort, L., Aronson, D., Counts, R.,
Edson, J., van Eys, J. et al. (1975) Proceedings: A
more uniform measurement of factor VIII inhibitors.
Thromb Diath Haemorrh 34: 612.
Kempton, C., Abshire, T., Deveras, R., Hoots, W.,
Gill, J., Kessler, C. et al. (2012) Pharmacokinetics
and safety of OBI-1, a recombinant B domain-deleted
porcine factor VIII, in subjects with haemophilia A.
Haemophilia 18: 798–804.
Kempton, C., Soucie, J., Miller, C., Hooper, C.,
Escobar, M., Cohen, A. et al. (2010) In non-severe
hemophilia A the risk of inhibitor after intensive factor
treatment is greater in older patients: a case-control
study. J Thromb Haemost 8: 2224–2231.
Kempton, C. and White, G., II, (2009) How we treat
a hemophilia A patient with a factor VIII inhibitor.
Blood 113: 11–17.
Konkle, B., Ebbesen, L., Erhardtsen, E., Bianco, R.,
Lissitchkov, T., Rusen, L. et al. (2007) Randomized,
prospective clinical trial of recombinant factor VIIa
for secondary prophylaxis in hemophilia patients with
inhibitors. J Thromb Haemost 5: 1904–1913.
Kono, H. and Rock, K. (2008) How dying cells alert
the immune system to danger. Nat Rev Immunol 8:
279–289.
Kroner, B. (1999) Comparison of the international
immune tolerance registry and the North American
immune tolerance registry. Vox Sang 77(Suppl. 1):
33–37.
Kurczynski, E. and Penner, J. (1974) Activated
prothrombin concentrate for patients with factor VIII
inhibitors. N Engl J Med 291: 164–167.
Kurnik, K., Bidlingmaier, C., Engl, W., Chehadeh, H.,
Reipert, B. and Auerswald, G. (2010) New early
prophylaxis regimen that avoids immunological
danger signals can reduce FVIII inhibitor
development. Haemophilia 16: 256–262.
Therapeutic Advances in Hematology 4 (1)
Lacroix-Desmazes, S., Dimitrov, J. and Repesse, Y.
(2009) Inhibitors of factor VIII in hemophilia. N Engl
J Med 361: 308; author reply 310.
Lee, C., Berntorp, E. and Hoots, W. (2005) Textbook
of Hemophilia. 1st edn. Malden, MA: Blackwell
Publishing.
Leissinger, C., Gringeri, A., Antmen, B.,
Berntorp, E., Biasoli, C., Carpenter, S. et al. (2011a)
Anti-inhibitor coagulant complex prophylaxis in
hemophilia with inhibitors. N Engl J Med 365:
1684–1692.
Leissinger, C., Kruse-Jarres, R., Granger, S.,
Konkle, B., Ragni, M., Journeycake, J. et al. (2011b)
Phase II trial of rituximab in the treatment of
inhibitors in congenital hemophilia A: results of the
RICH study. Blood (ASH Annual Meeting Abstracts)
118: 27.
Lenk, H. (2000) The German Registry of immune
tolerance treatment in hemophilia – 1999 update.
Haematologica 85(10 Suppl.): 45–47.
Ling, M., Duncan, E., Rodgers, S., Street, A. and
Lloyd, J. (2003) Low detection rate of antibodies to
non-functional epitopes on factor VIII in patients with
hemophilia A and negative for inhibitors by Bethesda
assay. J Thromb Haemost 1: 2548–2553.
Lollar, P. (2004) Pathogenic antibodies to coagulation
factors. Part one: factor VIII and factor IX. J Thromb
Haemost 2: 1082–1095.
Lorenzo, J., Lopez, A., Altisent, C. and Aznar, J.
(2001) Incidence of factor VIII inhibitors in severe
haemophilia: the importance of patient age. Br J
Haematol 113: 600–603.
Lozier, J., Rosenberg, P., Goedert, J. and Menashe, I.
(2011) A case-control study reveals immunoregulatory
gene haplotypes that influence inhibitor risk in severe
haemophilia A. Haemophilia 17: 641–649.
Lusher, J., Arkin, S., Abildgaard, C. and Schwartz, R.
(1993) Recombinant factor VIII for the treatment
of previously untreated patients with hemophilia
A. Safety, efficacy, and development of inhibitors.
Kogenate Previously Untreated Patient Study Group.
N Engl J Med 328: 453–459.
Lusher, J., Roberts, H., Davignon, G., Joist, J.,
Smith, H., Shapiro, A. et al. (1998) A randomized,
double-blind comparison of two dosage levels of
recombinant factor VIIa in the treatment of joint,
muscle and mucocutaneous haemorrhages in persons
with haemophilia A and B, with and without inhibitors.
rFVIIa Study Group. Haemophilia 4: 790–798.
Maclean, P., Richards, M., Williams, M., Collins, P.,
Liesner, R., Keeling, D. et al. (2011) Treatment
related factors and inhibitor development in children
with severe haemophilia A. Haemophilia 17:
282–287.
70 http://tah.sagepub.com
Manco-Johnson, M., Abshire, T., Shapiro, A., Riske, B.,
Hacker, M., Kilcoyne, R. et al. (2007) Prophylaxis
versus episodic treatment to prevent joint disease
in boys with severe hemophilia. N Engl J Med 357:
535–544.
Mannucci, P., Gringeri, A., Peyvandi, F. and
Santagostino, E. (2007) Factor VIII products and
inhibitor development: the SIPPET study (survey
of inhibitors in plasma-product exposed toddlers).
Haemophilia 13(Suppl. 5): 65–68.
Mannucci, P., Mancuso, M. and Santagostino,
E. (2012) How we choose factor VIII to treat
hemophilia. Blood 119: 4108–4114.
Mariani, G., Scheibel, E., Nogao, T., Kasper, C.,
Ewing, N., Mauser-Bunschoten, E. et al. (1994)
Immunetolerance as treatment of alloantibodies to
factor VIII in hemophilia. The International Registry
of Immunetolerance Protocols. Semin Hematol
31(2 Suppl. 4): 62–64.
Matzinger, P. (2002) The danger model: a renewed
sense of self. Science 296: 301–305.
Mauser-Bunschoten, E., Den Uijl, I., Schutgens, R.,
Roosendaal, G. and Fischer, K. (2012) Risk of
inhibitor development in mild haemophilia A
increases with age. Haemophilia 18: 263–267.
Meijer, P. and Verbruggen, B. (2009) The between-
laboratory variation of factor VIII inhibitor testing: the
experience of the external quality assessment program
of the ECAT foundation. Semin Thromb Hemost 35:
786–793.
Miao, C. (2010) Immunomodulation for inhibitors in
hemophilia A: the important role of Treg cells. Expert
Rev Hematol 3: 469–483.
Miller, C., Benson, J., Ellingsen, D., Driggers, J.,
Payne, A., Kelly, F. et al. (2012) F8 and F9 mutations
in US haemophilia patients: correlation with history of
inhibitor and race/ethnicity. Haemophilia 18: 375–382.
Morfini, M., Haya, S., Tagariello, G., Pollmann, H.,
Quintana, M., Siegmund, B. et al. (2007) European
study on orthopaedic status of haemophilia patients
with inhibitors. Haemophilia 13: 606–612.
Nilsson, I., Berntorp, E. and Zettervall, O. (1988)
Induction of immune tolerance in patients with
hemophilia and antibodies to factor VIII by combined
treatment with intravenous IgG, cyclophosphamide,
and factor VIII. N Engl J Med 318: 947–950.
Oldenburg, J., Picard, J., Schwaab, R., Brackmann, H.,
Tuddenham, E. and Simpson, E. (1997) HLA
genotype of patients with severe haemophilia A due
to intron 22 inversion with and without inhibitors of
factor VIII. Thromb Haemost 77: 238–242.
Oldenburg, J., Schroder, J., Brackmann, H., Muller-
Reible, C., Schwaab, R. and Tuddenham, E. (2004)

Environmental and genetic factors influencing inhibitor
development. Semin Hematol 41(1 Suppl. 1): 82–88.
Pavlova, A., Delev, D., Lacroix-Desmazes, S.,
Schwaab, R., Mende, M., Fimmers, R. et al.
(2009) Impact of polymorphisms of the major
histocompatibility complex class II, interleukin-10,
tumor necrosis factor-alpha and cytotoxic
T-lymphocyte antigen-4 genes on inhibitor
development in severe hemophilia A. J Thromb
Haemost 7: 2006–2015.
Peyvandi, F., Lotta, L. and Mannucci, P. (2009)
Inhibitors of factor VIII in hemophilia. N Engl J Med
361: 309; author reply 310.
Qadura, M., Othman, M., Waters, B., Chegeni, R.,
Walker, K., Labelle, A. et al. (2008) Reduction of the
immune response to factor VIII mediated through
tolerogenic factor VIII presentation by immature
dendritic cells. J Thromb Haemost 6: 2095–2104.
Qadura, M., Waters, B., Burnett, E., Chegeni, R.,
Bradshaw, S., Hough, C. et al. (2009) Recombinant
and plasma-derived factor VIII products induce
distinct splenic cytokine microenvironments in
hemophilia A mice. Blood 114: 871–880.
Ragni, M., Ojeifo, O., Feng, J., Yan, J., Hill, K.,
Sommer, S. et al. (2009a) Risk factors for inhibitor
formation in haemophilia: a prevalent case-control
study. Haemophilia 15: 1074–1082.
Ragni, M., Wu, W., Liang, X., Hsieh, C., Cortese-
Hassett, A. and Lu, L. (2009b) Factor VIII-pulsed
dendritic cells reduce anti-factor VIII antibody
formation in the hemophilia A mouse model. Exp
Hematol 37: 744–754.
Rawle, F., Pratt, K., Labelle, A., Weiner, H.,
Hough, C. and Lillicrap, D. (2006) Induction of
partial immune tolerance to factor VIII through prior
mucosal exposure to the factor VIII C2 domain.
J Thromb Haemost 4: 2172–2179.
Rivard, G., Lillicrap, D., Poon, M., Demers, C.,
Lepine, M., St-Louis, J. et al. (2005) Can activated
recombinant factor VII be used to postpone the
exposure of infants to factor VIII until after 2 years of
age? Haemophilia 11: 335–339.
Robbins, D., Kulkarni, R., Gera, R., Scott-Emuakpor, A.,
Bosma, K. and Penner, J. (2001) Successful treatment
of high titer inhibitors in mild hemophilia A with
avoidance of factor VIII and immunosuppressive
therapy. Am J Hematol 68: 184–188.
Saint-Remy, J., Lacroix-Desmazes, S. and Oldenburg,
J. (2004) Inhibitors in haemophilia: pathophysiology.
Haemophilia 10(Suppl. 4): 146–151.
Santagostino, E., Mancuso, M., Rocino, A.,
Mancuso, G., Mazzucconi, M., Tagliaferri, A.
et al. (2005) Environmental risk factors for inhibitor
http://tah.sagepub.com 71
C Witmer and G Young
development in children with haemophilia A: a case-
control study. Br J Haematol 130: 422–427.
Santos, A., Annichino-Bizzacchi, J. and Ozelo, M.
(2009) Inhibitors of factor VIII in hemophilia. N Engl
J Med 361: 309–310; author reply 310.
Scandella, D., Mattingly, M., de Graaf, S. and
Fulcher, C. (1989) Localization of epitopes for human
factor VIII inhibitor antibodies by immunoblotting
and antibody neutralization. Blood 74: 1618–1626.
Scharrer, I., Bray, G. and Neutzling, O. (1999)
Incidence of inhibitors in haemophilia A patients – a
review of recent studies of recombinant and plasma-
derived factor VIII concentrates. Haemophilia 5:
145–154.
Schneiderman, J., Nugent, D. and Young, G. (2004)
Sequential therapy with activated prothrombin
complex concentrate and recombinant factor VIIa
in patients with severe haemophilia and inhibitors.
Haemophilia 10: 347–351.
Shapiro, A., Gilchrist, G., Hoots, W., Cooper, H. and
Gastineau, D. (1998) Prospective, randomised trial
of two doses of rFVIIa (NovoSeven) in haemophilia
patients with inhibitors undergoing surgery. Thromb
Haemost 80: 773–778.
Sharathkumar, A., Lillicrap, D., Blanchette, V.,
Kern, M., Leggo, J., Stain, A. et al. (2003) Intensive
exposure to factor VIII is a risk factor for inhibitor
development in mild hemophilia A. J Thromb Haemost
1: 1228–1236.
Sjamsoedin, L., Heijnen, L., Mauser-Bunschoten, E.,
van Geijlswijk, J., van Houwelingen, H., van Asten,
P. et al. (1981) The effect of activated prothrombin-
complex concentrate (FEIBA) on joint and muscle
bleeding in patients with hemophilia A and antibodies
to factor VIII. A double-blind clinical trial. N Engl J
Med 305: 717–721.
Soucie, J., Evatt, B. and Jackson, D. (1998)
Occurrence of hemophilia in the United States. The
Hemophilia Surveillance System Project Investigators.
Am J Hematol 59: 288–294.
Su, R., Epp, A., Latchman, Y., Bolgiano, D.,
Pipe, S. and Josephson, N. (2010) Suppression of
FVIII inhibitor formation in hemophilic mice by
delivery of transgene modified apoptotic fibroblasts.
Mol Ther 18: 214–222.
Sultan, Y. (1992) Prevalence of inhibitors in a
population of 3435 hemophilia patients in France.
French Hemophilia Study Group. Thromb Haemost
67: 600–602.
ter Avest, P., Fischer, K., Mancuso, M., Santagostino, E.,
Yuste, V., van den Berg, H. et al. (2008) Risk stratification
for inhibitor development at first treatment for severe
hemophilia A: a tool for clinical practice. J Thromb
Haemost 6: 2048–2054.
Therapeutic Advances in Hematology 4 (1)
van der Bom, J., Mauser-Bunschoten, E., Fischer, K.
and van den Berg, H. (2003) Age at first treatment
and immune tolerance to factor VIII in severe
hemophilia. Thromb Haemost 89: 475–479.
Verbruggen, B., Novakova, I., Wessels, H.,
Boezeman, J., van den Berg, M. and Mauser-
Bunschoten, E. (1995) The Nijmegen modification
of the Bethesda assay for factor VIII:C inhibitors:
improved specificity and reliability. Thromb Haemost
73: 247–251.
Viel, K., Ameri, A., Abshire, T., Iyer, R., Watts, R.,
Lutcher, C. et al. (2009) Inhibitors of factor VIII in black
patients with hemophilia. N Engl J Med 360: 1618–1627.
Vlot, A., Wittebol, S., Strengers, P., Turenhout, E.,
Voorberg, J., van den Berg, H. et al. (2002) Factor
VIII inhibitor in a patient with mild haemophilia A
and an Asn618-->Ser mutation responsive to immune
tolerance induction and cyclophosphamide. Br J
Haematol 117: 136–140.
Waters, B. and Lillicrap, D. (2009) The molecular
mechanisms of immunomodulation and tolerance
induction to factor VIII. J Thromb Haemost 7: 1446–1456.
Visit SAGE journals online White, G., Rosendaal, F. 2nd, Aledort, L., Lusher, J.,
http://tah.sagepub.com
Rothschild, C. and Ingerslev, J. (2001) Definitions
SAGE journals in hemophilia. Recommendation of the scientific
72 http://tah.sagepub.com
subcommittee on factor VIII and factor IX of the
scientific and standardization committee of the
International Society on Thrombosis and Haemostasis.
Thromb Haemost 85: 560.
Wight, J. and Paisley, S. (2003) The epidemiology
of inhibitors in haemophilia A: a systematic review.
Haemophilia 9: 418–435.
Yang, G., Yao, L. and Lu, Z. (2009) Inhibitors of
factor VIII in hemophilia. N Engl J Med 361: 309;
author reply 310.
Young, G., Auerswald, G., Jimenez-Yuste, V.,
Konkle, B., Lambert, T., Morfini, M. et al. (2011)
When should prophylaxis therapy in inhibitor
patients be considered? Haemophilia 17:
e849–e857.
Young, M., Inaba, H., Hoyer, L., Higuchi, M.,
Kazazian, H., Jr, and Antonarakis, S. (1997)
Partial correction of a severe molecular defect in
hemophilia A, because of errors during expression
of the factor VIII gene. Am J Hum Genet 60:
565–573.
Zakarija, A., Harris, S., Rademaker, A., Brewer,
J., Krudysz-Amblo, J., Butenas, S. et al. (2011)
Alloantibodies to factor VIII in haemophilia.
Haemophilia 17: 636–640.