The n e w e ng l a n d j o u r na l of m e dic i n e

original article

Factor VIII Products and Inhibitor

Development in Severe Hemophilia A
Samantha C. Gouw, M.D., Ph.D., Johanna G. van der Bom, M.D., Ph.D.,
Rolf Ljung, M.D., Ph.D., Carmen Escuriola, M.D., Ana R. Cid, M.D.,
Ségolène Claeyssens-Donadel, M.D., Christel van Geet, M.D., Ph.D.,
Gili Kenet, M.D., Anne Mäkipernaa, M.D., Ph.D., Angelo Claudio Molinari, M.D.,
Wolfgang Muntean, M.D., Rainer Kobelt, M.D., George Rivard, M.D.,
Elena Santagostino, M.D., Ph.D., Angela Thomas, M.D., Ph.D.,
and H. Marijke van den Berg, M.D., Ph.D.,
for the PedNet and RODIN Study Group*

A bs t r ac t

Background
For previously untreated children with severe hemophilia A, it is unclear whether The authors’ affiliations are listed in the
the type of factor VIII product administered and switching among products are as- Appendix. Address reprint requests to
Dr. van den Berg at the University Medi-
sociated with the development of clinically relevant inhibitory antibodies (inhibitor cal Center Utrecht, Julius Center for
development). Health Sciences and Primary Care, Rm.
No. Stratenum 5.125, P.O. Box 85500,
Methods 3508 GA Utrecht, the Netherlands, or at
We evaluated 574 consecutive patients with severe hemophilia A (factor VIII activity, h.m.vandeberg@umcutrecht.nl.
<0.01 IU per milliliter) who were born between 2000 and 2010 and collected data on * Investigators in the European Pediatric
all clotting-factor administration for up to 75 exposure days. The primary outcome Network for Hemophilia Management
was inhibitor development, which was defined as at least two positive inhibitor tests (PedNet) and the Research of Deter­
minants of Inhibitor Development
with decreased in vivo recovery of factor VIII levels. (RODIN) study group are listed in the
Supplementary Appendix, available at
Results
NEJM.org.
Inhibitory antibodies developed in 177 of the 574 children (cumulative incidence,
N Engl J Med 2013;368:231-9.
32.4%); 116 patients had a high-titer inhibitory antibody, defined as a peak titer of at DOI: 10.1056/NEJMoa1208024
least 5 Bethesda units per milliliter (cumulative incidence, 22.4%). Plasma-derived Copyright © 2013 Massachusetts Medical Society.
products conferred a risk of inhibitor development that was similar to the risk with
recombinant products (adjusted hazard ratio as compared with recombinant prod-
ucts, 0.96; 95% confidence interval [CI], 0.62 to 1.49). As compared with third-
generation full-length recombinant products (derived from the full-length comple-
mentary DNA sequence of human factor VIII), second-generation full-length prod-
ucts were associated with an increased risk of inhibitor development (adjusted
hazard ratio, 1.60; 95% CI, 1.08 to 2.37). The content of von Willebrand factor in
the products and switching among products were not associated with the risk of
inhibitor development.
Conclusions
Recombinant and plasma-derived factor VIII products conferred similar risks of
inhibitor development, and the content of von Willebrand factor in the products
and switching among products were not associated with the risk of inhibitor devel-
opment. Second-generation full-length recombinant products were associated with
an increased risk, as compared with third-generation products. (Funded by Bayer
Healthcare and Baxter BioScience.)

n engl j med 368;3  nejm.org  january 17, 2013 231

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 The n e w e ng l a n d j o u r na l
P
atients with severe hemophilia A
have a deficiency of functional clotting fac-
tor VIII (<0.01 IU per milliliter) and have
bleeding in the joints and muscles. To prevent
joint destruction, the current standard of care for
children with severe hemophilia A is primary pro-
phylaxis. This includes regular infusions of fac-
tor VIII, which are initiated at the time of the first
episode of bleeding in a joint or earlier, aiming at
the prevention of joint damage.1 However, in about
30% of children, inhibitory antibodies to infused
factor VIII products develop, making usual treat-
ment with factor VIII and prophylaxis impossible.
There are multiple risk factors for the develop-
ment of inhibitory antibodies (inhibitor develop-
ment).2-18
It has been suggested that recombinant factor
VIII products are more immunogenic than plasma-
derived products. However, the outcomes of nu-
merous studies and systematic reviews have
been contradictory.19-23 The studies have been
limited by the enrollment of small, heteroge-
neous study populations and the use of several
factor VIII products, and comparisons among
studies have been difficult because of different
study designs.22,24 The inclusion of minimally
treated patients and patients who were still at risk
for subsequent development of inhibitory anti-
bodies has led to an underestimation of the in-
cidence of inhibitor development.25 In addition,
prospective postmarketing studies could not in-
clude high-risk children who started bleeding at
an early age, which meant that the risk of in-
hibitor development was underestimated. Fur-
thermore, small studies with extreme results are
more likely to be published than are those with
less extreme findings.22 For these reasons, three
systematic reviews of the immunogenicity of
factor VIII products resulted in different conclu-
sions.21-23 Randomized trials comparing the im-
munogenicity of factor VIII products have not yet
been completed.26
A finding that recombinant and plasma-
derived products had a differential risk with re-
spect to inhibitor development would influence
both the decision about which type of product to
administer in individual patients and the avail-
ability of the preferred product. Therefore,
knowledge of the risk of inhibitor development
associated with recombinant and plasma-derived
products is important for both the individual
patient with hemophilia and the hemophilia
population as a whole. We assessed whether the
232 n engl j med 368;3  nejm.org  january 17, 2013
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of m e dic i n e
type of factor VIII product and switching among
products were associated with inhibitor develop-
ment in previously untreated children with severe
hemophilia A.
Me thods
Patients
We enrolled consecutive, previously untreated pa-
tients with severe hemophilia A (factor VIII activ-
ity, <0.01 IU per milliliter) that had been diag-
nosed in 1 of the 29 participating hemophilia
treatment centers. All the children in the study
were born between January 1, 2000, and January
1, 2010. Children who had been referred to the
centers because of the presence of inhibitory an-
tibodies were excluded from the study. Approval
was obtained from the institutional review board
at each study center. Parents or guardians of all
children provided written informed consent.
Data Collection
We uniformly collected detailed data on all infu-
sions of factor VIII for up to 75 exposure days or
until the development of inhibitory antibodies,
including dates of infusion, doses and brands of
factor VIII products, reasons for treatment, types
of bleeding, extravasation of products, and surgery.
Patients were followed until the development
of a clinically relevant inhibitory antibody or a
cumulative number of 75 exposure days. (After
75 exposure days, inhibitor development becomes
rare [approximately 2 to 5 cases per 1000 patient-
years]).27
Outcomes
The primary outcome was the development of
clinically relevant inhibitory antibodies, which
was defined as at least two positive inhibitor ti-
ters combined with decreased in vivo recovery of
factor VIII levels up to the 75th exposure day. The
secondary outcome was the development of a
high-titer inhibitor, which was defined as a peak
titer of at least 5 Bethesda units per milliliter up
to the 75th exposure day.28 A positive inhibitor
titer was defined according to the cutoff level of
the inhibitor assay used in the laboratory at each
center. Factor VIII recovery was described as de-
creased if the level of factor VIII activity was less
than 66% of the expected level 15 minutes after
the infusion of factor VIII. The expected level of
factor VIII activity was calculated according to the
criteria of Lee et al.29
 Development of Factor VIII Inhibitors in Hemophilia A
In the majority of centers (92%), patients were
routinely screened for inhibitor development af-
ter every 1 to 5 exposure days during the first 20
exposure days and at least every 3 months there-
after. At all centers, patients were closely moni-
tored for signs of inhibitor development, and
investigators performed inhibitor and recovery
testing if there was any suspicion that inhibitory
antibodies had developed.
Types of Factor VIII Products
We assessed the incidence of inhibitor develop-
ment according to the type of product used at
subsequent exposure days (time-varying determi-
nant). We categorized factor VIII products in sev-
eral ways. First, we compared the inhibitor risk
between plasma-derived factor VIII products and
recombinant products. Second, to investigate
whether the content of von Willebrand factor was
associated with the risk of inhibitor development,
we categorized factor VIII products into products
containing no von Willebrand factor (all recom-
binant products), products containing less than
0.01 IU of von Willebrand factor antigen per inter-
national unit of factor VIII antigen (monoclonal
antibody–purified plasma-derived products), and
products containing 0.01 IU or more of von Will-
ebrand factor per international unit of factor VIII
antigen (other plasma-derived products).30 Third,
we compared inhibitor incidence among the fol-
lowing categories of factor VIII products: plasma-
derived products, first-generation full-length re-
combinant product (derived from the full-length
complementary DNA sequence of human factor
VIII) (Recombinate, Baxter BioScience), second-
generation B-domain–deleted recombinant prod-
uct, and second- and third-generation full-length
recombinant products.
We did not evaluate Kogenate (Bayer Health-
care), a first-generation full-length recombinant
product, or Refacto AF (Pfizer), a third-generation
B-domain–deleted product, because of the small
numbers of patients who received these products
(10 patients [7 as first-use product] and 3 pa-
tients [3 as first-use product], respectively). The
product type that was used most frequently was
selected as the reference category.
Switching among Products
We evaluated the risk of inhibitor development in
children who were receiving a plasma-derived
product who were then switched to a recombi-
nant product, as compared with those who were
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still receiving a plasma-derived product. We sim-
ilarly assessed the association between switching
among various types of factor VIII products and
the development of inhibitory antibodies.
Study Conduct
The study was supported by unrestricted research
grants from Bayer Healthcare and Baxter Bio­
Science. The companies did not have a role in the
study design, data collection, data analysis, or writ-
ing of the manuscript. Representatives of the com-
panies reviewed the manuscript before it was sub-
mitted for publication. No one who is not an author
contributed to the writing of the manuscript.
Three of the authors (including the first au-
thor) designed the study, performed statistical
analyses, interpreted the data, and vouch for the
integrity of the data, the fidelity of the study to
the protocol, and the accuracy of the data analyses.
The first author wrote the first draft of the manu-
script. All the other authors collected the data,
critically reviewed the manuscript, and made the
decision to submit the manuscript for publication.
Statistical Analysis
The absolute risk of inhibitor development varies
according to the cumulative number of exposure
days. To account for this varying risk, we used
pooled logistic regression with the cumulative
number of exposure days as the time variable in-
stead of calendar time. We pooled observations
over all exposure days for all patients into a sin-
gle sample and then used a logistic-regression
model with stratification according to number of
exposure days to relate the risk factors to inhibitor
development. This method accounts for varying
risks according to the cumulative number of ex-
posure days and is equivalent to Cox regression
with exposure days as time-variable and time-
dependent covariates.31 Relative hazard rates
were interpreted as relative risks.
We calculated both unadjusted and adjusted
hazard ratios, with the latter adjusted for race or
ethnic group; age at first exposure to factor VIII;
reason for first treatment; interval between ex-
posure days; dose of factor VIII; F8 genotype;
and status with respect to family history of he-
mophilia and inhibitors, history of switching
among product brands, peak treatment episodes
(defined as treatment with factor VIII for bleed-
ing or for surgery on either ≥3 consecutive days
or ≥5 consecutive days), a history of major sur-
gery, and regular prophylaxis. Coding details are
233
 The n e w e ng l a n d j o u r na l of m e dic i n e

efficacy) for previously untreated patients. An

648 Patients were eligible
25 Were excluded
5 Did not provide informed consent
10 Did not have available data
8 Were lost to follow-up
1 Died from intracranial hemorrhage
1 Had unknown reason
17 Had pending informed consent
606 Were included
22 Had baseline-only data
6 Were not yet treated
3 Were lost to follow-up
8 Had data of insufficient quality
5 Had unspecified reasons
7 Had no inhibitor development
15 Had unknown inhibitor status
independent statistician who was unaware of
product types repeated all results by means of
Cox proportional-hazards regression models.
R e sult s
Patients
A total of 648 patients were eligible for the study.
Of these, 17 patients were excluded because of
pending informed consent, and 25 patients were
excluded by the investigators for various other
reasons (Fig. 1). Baseline data were available for
the remaining 606 patients; the analysis included
574 of these patients (94.7%), for whom detailed
exposure data were available. Their characteris-
tics according to the type of factor VIII product
that was first used are presented in Table 1 (see
also Table 1S in the Supplementary Appendix).

584 Remained in study Primary Outcome

8 Had insufficient data on exposure days
(reached study end point)
2 Had inhibitor development
6 Had no inhibitor development
2 Received unknown factor VIII product
at first exposure
574 Remained in study
Clinically relevant inhibitory antibodies developed
in 177 patients (cumulative incidence, 32.4%; 95%
confidence interval [CI], 28.5 to 36.3). Of these
patients, 116 had high-titer inhibitors (cumula-
tive incidence, 22.4%; 95% CI, 18.8 to 26.0). In-
hibitory antibodies developed after a median of
15 exposure days (interquartile range, 10 to 20)
at a median age of 15.5 months (interquartile
range, 10.7 to 19.6).

Plasma-Derived versus Recombinant Products

516 Reached study end point
58 Did not reach study end point
47 Had treatment data updated until
Jan. 9, 2010
11 Did not have treatment data updated
3 Moved to another center
2 Died from intracranial hemorrhage
1 Was lost to follow-up
5 Had unspecified reasons
Figure 1. Enrollment and Outcomes.
provided in the Supplementary Appendix, avail-
able with the full text of this article at NEJM.org.
To assess whether our findings were robust,
we also compared the incidence of inhibitor de-
velopment according to the product brands used
at the first exposure to factor VIII (a time-fixed
determinant). We repeated the analyses in the
subgroup of patients who were not included in
registration trials (primary studies of safety and
Plasma-derived products were used on 4018 expo-
sure days, and recombinant products were used
on 25,661 exposure days. Plasma-derived products
carried a risk of inhibitor development that was
similar to the risk with recombinant products (ad-
justed hazard ratio as compared with recombinant
products, 0.96; 95% CI, 0.62 to 1.49) (Table 2 and
Fig. 2).
Content of von Willebrand Factor
Seven patients received products with a low von
Willebrand factor content on 1 to 11 exposure
days (total, 26 exposure days). Inhibitory anti-
bodies developed in two patients after receiving a
product with a low von Willebrand factor content.
The risk of inhibitor development with products
containing a high amount of von Willebrand fac-
tor was similar to the risk with products contain-
ing no von Willebrand factor (adjusted hazard
ratio, 0.90; 95% CI, 0.57 to 1.41).

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 Table 1. Characteristics of the Patients and the Type of Factor VIII Product Administered during the First Treatment.

Plasma-Derived All Product Types

Characteristic Recombinant Product* Product (N = 88) (N = 574)
Third-Generation Second-Generation First-Generation Second-Generation
Full-Length Full-Length Full-Length B-Domain–Deleted
(N = 157) (N = 183) (N = 59) (N = 77)
Patients who switched product brands — no. (%) 4 (2.5) 16 (8.7) 25 (42.4) 15 (19.5) 28 (31.8) 94 (16.4)
Patients who switched from plasma-derived to 0 2 (1.1) 0 1 (1.3) 17 (19.3) 20 (3.5)
recombinant products — no. (%)
Median age (interquartile range) — yr 4.6 (3.5–6.5) 6.1 (3.8–8.5) 9.3 (8.4–10.8) 9.1 (5.7–10.5) 6.4 (4.2–9.6) 6.4 (4.0–8.9)
White race — no. (%)† 137 (87.3) 167 (91.3) 54 (91.5) 73 (94.8) 81 (92.0) 521 (90.8)
Family history of hemophilia — no. (%)
No 71 (45.2) 117 (63.9) 27 (45.8) 42 (54.5) 44 (50.0) 304 (53.0)
Yes
Negative for inhibitors 64 (40.8) 50 (27.3) 21 (35.6) 21 (27.3) 24 (27.3) 187 (32.6)
Positive for inhibitors 22 (14.0) 16 (8.7) 11 (18.6) 14 (18.2) 20 (22.7) 83 (14.5)
F8 genotype — no. (%)‡
High-risk 95 (60.5) 100 (54.6) 35 (59.3) 37 (48.1) 56 (63.6) 331 (57.7)
Low-risk 45 (28.7) 60 (32.8) 18 (30.5) 20 (26.0) 28 (31.8) 172 (30.0)
Median age at first exposure to factor VIII (interquar- 9.9 (5.3–13.5) 10.2 (7.5–14.1) 9.7 (3.8–11.6) 8.8 (2.9–14.0) 7.9 (3.7–12.8) 9.8 (5.4–13.5)
tile range) — mo
Initiation of regular prophylaxis within first 50 expo- 110 (70.1) 125 (68.3) 40 (67.8) 62 (80.5) 67 (76.1) 411 (71.6)
sure days — no. (%)§
Median cumulative exposure days at start of prophy- 17.0 (9.8–25.3) 12.0 (5.0–22.5) 18.0 (11.3–31.8) 18.5 (8.0–26.3) 12 (5–21) 15 (7–25)

n engl j med 368;3  nejm.org  january 17, 2013

The New England Journal of Medicine

laxis (interquartile range) — no.§
History of peak treatment episode on first exposure
day — no. (%)¶
≥3 days 40 (25.5) 41 (22.4) 15 (25.4) 20 (26.0) 30 (34.1) 149 (26.0)
Development of Factor VIII Inhibitors in Hemophilia A

≥5 days 28 (17.8) 21 (11.5) 9 (15.3) 14 (18.2) 24 (27.3) 98 (17.1)

Copyright © 2013 Massachusetts Medical Society. All rights reserved.

History of surgical procedure — no. (%) 46 (29.3) 33 (18.0) 18 (30.5) 28 (36.4) 16 (18.2) 144 (25.1)
Inhibitor development — no. (%)‖
Clinically relevant 41 (28.2) 64 (37.7) 17 (29.0) 23 (30.3) 29 (33.1) 177 (32.4)
High-titer 25 (17.9) 40 (25.2) 14 (24.6) 13 (18.0) 21 (25.7) 116 (22.4)

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* Among the recombinant products, data are not included for seven patients who were first treated with Kogenate (Bayer Healthcare), a first-generation full-length product, and three patients
who were first treated with Refacto AF (Wyeth), a third-generation B-domain–deleted product. However, these patients are included in the total number of patients who received any product.
† Race was self-reported.
‡ Low-risk genotypes included those with small deletions and insertions, missense mutations, and splice-site mutations. High-risk genotypes included those with large deletions, non-
sense mutations, and intron 1 and 22 inversions.
§ The start of regular prophylaxis was defined as the moment at which at least three consecutive prophylactic infusions of factor VIII had been given within a period of at least 15 days.
¶ A peak treatment episode was defined as treatment with factor VIII for bleeding or for surgery on either at least 3 consecutive days or at least 5 consecutive days.
‖ The values for inhibitor development are cumulative incidences, which were calculated by means of the Kaplan–Meier method.

235
 The n e w e ng l a n d j o u r na l of m e dic i n e

Specific Product Types

P Value Hazard Ratio† P Value

‡ A first-generation full-length recombinant product, Kogenate (Bayer Healthcare), and a third-generation B-domain–deleted recombinant product, Refacto AF (Wyeth), were used only in
The risk of inhibitor development was similar

0.85

0.02
0.53
0.92
0.51

and status with respect to family history of hemophilia and inhibitors, history of switching among product brands, peak treatment episodes of either at least 3 consecutive days or at
NA
NA

† Hazard ratios were adjusted for race or ethnic group; age at first exposure to factor VIII; reason for first treatment; interval between exposure days; dose of factor VIII; F8 genotype;
among plasma-derived products, first-generation

1.79 (1.09–2.94)
1.26 (0.61–2.61)
0.97 (0.49–1.91)
1.23 (0.67–2.28)
0.95 (0.56–1.61)
full-length recombinant products, second-gener-
Adjusted

1.00 ation B-domain–deleted recombinant products,

1.00
and third-generation recombinant products. First-
High-Titer Inhibitor Development

generation recombinant products were associat-

ed with an unadjusted hazard ratio of 1.44 (95%
CI, 0.71 to 2.90) for high-titer inhibitor develop-
0.40

0.12
0.31
0.82
0.17
NA

NA
ment; however, after adjustment, the hazard ra-
tio was lower. Second-generation full-length re-
Unadjusted Hazard

combinant products were associated with a

1.24 (0.75–2.03)

1.47 (0.91–2.38)
1.44 (0.71–2.90)
0.93 (0.48–1.79)
1.51 (0.84–2.71)
significantly higher risk of inhibitor develop-
Ratio

1.00

1.00

ment than were third-generation products (ad-

10 and 3 patients on 103 and 163 exposure days, respectively, so the effect of these products on inhibitor development was not studied.
justed hazard ratio, 1.60; 95% CI, 1.08 to 2.37;
P = 0.02); for high-titer inhibitor development,
the adjusted hazard ratio was 1.79 (95% CI, 1.09
Exposure Days

to 2.94; P = 0.02) (Table 2).

25,661
4,018

9,297
9,143
2,464
4,491
4,018
No. of

Switching among Products

Details about the analyses of switching among
brands of factor VIII are provided in the Supple-
Hazard Ratio P Value Hazard Ratio† P Value

0.87

0.02
0.96
0.97
0.56
NA

NA

mentary Appendix. Switching among products

was not associated with the risk of inhibitor de-
1.60 (1.08–2.37)
0.99 (0.53–1.83)
1.01 (0.60–1.70)
1.16 (0.70–1.92)
0.96 (0.62–1.49)

velopment (adjusted hazard ratio as compared

§ The only first-generation full-length product in this category was Recombinate (Baxter BioScience).
Adjusted

with no switching, 0.99; 95% CI, 0.63 to 1.56)

1.00

1.00

(Table 3S in the Supplementary Appendix).

Any Inhibitor Development
Table 2. Risk of Inhibitor Development, According to the Type of Factor VIII Product.*

Sensitivity Analyses
0.54

0.11
0.72
0.99
0.27

The results of sensitivity analyses regarding the

NA

NA

use of factor VIII products (plasma-derived vs.

least 5 consecutive days, history of major surgery, and regular prophylaxis.
1.14 (0.75–1.72)

1.37 (0.93–2.01)
1.12 (0.61–2.04)
1.00 (0.60–1.65)
1.31 (0.81–2.11)

recombinant products and specific product types)

Unadjusted

were similar to those of the primary analysis.

1.00

1.00

Details regarding the sensitivity analyses are pro-

vided in the Supplementary Appendix.
Exposure Days

Discussion
No. of

25,661
4,018

9,297
9,143
2,464
4,491
4,018

In this cohort study involving 574 consecutive,

previously untreated children with severe hemo-
philia A who were born between 2000 and 2010,
Second-generation B-domain–deleted
All recombinant vs. all plasma-derived

recombinant factor VIII products conferred a risk

of inhibitor development that was similar to the
Second-generation full-length
First-generation full-length§
Third-generation full-length

risk conferred by plasma-derived products. The

* NA denotes not applicable.

von Willebrand factor content in factor VIII prod-

ucts was not associated with inhibitor develop-
ment. Second-generation full-length recombinant
Specific products
products

products were associated with a higher risk of

Plasma-derived

Plasma-derived
Recombinant‡
Recombinant

inhibitor development than were third-generation

full-length products. Switching from a plasma-
Product

derived product to a recombinant product or

switching among brands of factor VIII products

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 Development of Factor VIII Inhibitors in Hemophilia A

did not result in an increased risk of inhibitor

3
development.

Adjusted Relative Risk (95% CI)

We directly compared the use of recombinant
products and plasma-derived products in one
study cohort. We avoided selection bias by in- 2

cluding all consecutive patients who were born

between January 1, 2000, and January 1, 2010.
We excluded all patients who were referred from 1
nonparticipating hemophilia centers because of
inhibitor development. We used survival analysis
because at the moment of data analysis a num- 0
ber of patients had not yet reached the study end

nt

ed

th

th

ed
te
gt
na

ng

ng
iv

iv
ele
en
point and were still at risk for inhibitor develop-

er

er
bi

Le

Le

D
D

D
m

ll-

ll-

ll-

n–
a-

a-
o

Fu

Fu
ment. This enabled us to include all patients up

m
ec

ai
s

as
lR

om
la

io

io

tio

Pl
Al

lP
to the last exposure day and to calculate cumula-

at

at

D
ra
Al

B-
er

er

e
en

en

en

n
tive incidences. Collection of detailed informa-

io
G

at
d

t
1s

er
3r

2n
tion on all 75 exposure days allowed us to adjust

en
G
the associations for potential confounding fac-

tors. These findings were robust in sensitivity
analyses.
Even though we adjusted for potentially con-
founding factors, we cannot rule out residual
confounding. The observed associations may have
been affected by information bias, if frequencies
and methods of inhibitor screening among cen-
ters had differed according to the particular
factor VIII product. However, we would not ex-
pect that the cumulative incidence of high-titer
inhibitor development would be influenced by
variations in inhibitor screening because of a lack
of central laboratory testing, since these inhibi-
tory antibodies will always be detected clinically.
Since the associations were similar with respect
to both all clinically relevant inhibitor develop-
ment and high-titer inhibitor development, in-
formation bias would therefore not have played
a major role. Because a relatively small number
of patients were treated with plasma-derived
products and because of the variety of plasma-
derived products, we may not have been able to
detect potential differences in the risk of inhibi-
tor development among various plasma-derived
products.
Several reports have suggested that plasma-
derived factor VIII products, especially those con-
taining considerable amounts of von Willebrand
factor, are less immunogenic than recombinant
products.19,32,33 However, several systematic re-
views have yielded inconclusive results.21-23 Our
results are in agreement with the findings of a
similarly designed study, the Concerted Action on
Neutralizing Antibodies in Severe Hemophilia A
d
2n
Figure 2. Adjusted Relative Risk of Inhibitor Development, According to
the Type of Factor VIII Product.
Data are for 574 previously untreated children with severe hemophilia A. In
the comparison between all recombinant products and all plasma-derived
products (at left), the reference group was the recombinant products. In
the comparison of specific products (at right), the reference group was
third-generation full-length recombinant products. The only first-generation
full-length recombinant product that was evaluated was Recombinate (Baxter
BioScience). The I bars indicate 95% confidence intervals.
(CANAL) study, in which the risk of inhibitor
development was not clearly lower with plasma-
derived products than with recombinant prod-
ucts (relative risk, 0.79; 95% CI, 0.49 to 1.28).20
Unexpectedly, the risk of inhibitor develop-
ment was 60% higher among children receiving
a second-generation full-length recombinant
product than among those receiving a third-
generation full-length product. This association
may be a biased finding (through confounding,
selection bias, or information bias), a chance find-
ing, or a causal effect.
We accounted for bias from confounding by
adjusting the association for multiple potential
confounding factors. We summarized potentially
confounding factors according to the product
type used at the first treatment (Table 1). Chil-
dren at increased risk for inhibitor development
did not receive second-generation full-length fac-
tor VIII products more often than they did third-
generation products. Therefore, confounding does
not seem to explain this association.
We avoided selection bias by including all con-

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Copyright © 2013 Massachusetts Medical Society. All rights reserved.
 The n e w e ng l a n d j o u r na l of m e dic i n e

secutive patients and by excluding all patients who
were referred to the participating center because
of a known increased risk for inhibitors. In addi-
tion, to further confirm the absence of selection
bias, we performed a sensitivity analysis among
patients who were not in a safety and efficacy
trial. Patients who were included in such a trial
might have been at reduced risk for inhibitor de-
velopment, since they did not have early bleeding.
The observed increase in the risk of inhibitor
development with second-generation full-length
recombinant products as compared with third-
generation full-length products is not likely to be
affected by information bias, since it is unlikely
that patients who were treated with a second-
generation full-length product were more often
screened for inhibitors than those treated with a
third-generation product. Furthermore, we ob-
served a similar association in high-titer develop-
ment. Thus, selection bias and information bias
do not explain the observed increase in risk in
second-generation full-length recombinant prod-
ucts, as compared with third-generation full-
length products.
This difference in risk between recombinant
products may be due to chance, which seems un-
likely, given the precision of our estimate of effect.
But as long as the observation in our study is not
confirmed in other studies, we cannot exclude
the possibility. However, since bias is unlikely
and the probability of a chance finding is low,
the observed increase in the risk of inhibitor
development in patients receiving second-genera-
tion full-length factor VIII products may be real.
Other studies — including a systematic re-
view23 and reports of the Kogenate Bayer Study
Group25,34 — have not shown significant differ-
ences in the risk of inhibitor development
among various recombinant factor VIII products.
In the registration studies, the incidence of in-
hibitor development may have been underesti-
mated because of the inclusion of patients who
had already been treated with factor VIII on sev-
eral exposure days and a short follow-up period
for the subgroup of patients who were still at
risk for inhibitor development. There is no
straightforward biologic explanation for a dif-
ference in immunogenicity among recombinant
factor VIII products. Further studies are needed
to verify these observations and to identify bio-
logic explanations.
In conclusion, the use of recombinant factor
VIII products in children with severe hemophilia A
did not have a significant effect on the risk of
inhibitor development, as compared with the use
of plasma-derived products, nor was the von
Willebrand factor content of the products or
switching among them associated with the risk
of inhibitor development. An unexpected find-
ing was that second-generation full-length re-
combinant products were associated with an
increased risk of inhibitor development, as
compared with third-generation products.
Supported by unrestricted research grants from Bayer
Healthcare and Baxter BioScience.
Disclosure forms provided by the authors are available with
the full text of this article at NEJM.org.
We thank the study coordinator, Ella Smink; Emma Smid and
Mojtaba Hashemi for their support in data cleaning; Yves Guil-
laume, Kate Khair, Karin Lindvall, Monique Spoor, and Bep
Verkerk for their assistance in the study; and J. Michael Soucie
(Division of Blood Disorders, National Center on Birth Defects
and Developmental Disabilities, Centers for Disease Control and
Prevention) for repeating the analyses.

Appendix
The authors’ affiliations are as follows: the Department of Pediatrics, Wilhelmina Children’s Hospital (S.C.G.), and the Julius Center
for Health Sciences and Primary Care, University Medical Center Utrecht (S.C.G., H.M.B.), Utrecht, and the Department of Clinical
Epidemiology, Leiden University Medical Center, and the Center for Clinical Transfusion Research, Sanquin Foundation, Leiden (J.G.B.)
— all in the Netherlands; Lund University, Department of Pediatrics and Malmö Center for Thrombosis and Hemostasis, Skånes Uni-
versitetssjukhus, Malmö, Sweden (R.L.); Department of Pediatrics, J.W. Goethe University Hospital, Frankfurt, Germany (C.E.); Unidad
de Hemostasia y Trombosis, Hospital Universitario y Politécnico La Fe, Valencia, Spain (A.R.C.); Centre Regional d’Hemophilie, Centre
Hospitalier Universitaire, Toulouse, France (S.C.-D.); the Department of Pediatrics, University Hospitals Leuven, and the Department of
Cardiovascular Sciences, KU Leuven — both in Leuven, Belgium (C.G.); National Hemophilia Center, Ministry of Health, Sheba Medical
Center, Tel Hashomer, Israel (G.K.); Hospital for Children and Adolescents, University of Helsinki, Helsinki (A.M.); Dipartimento di
Ematologia ed Oncologia, Unità Trombosi ed Emostasi, Ospedale Pediatrico Giannina Gaslini, Genoa (A.C.M.), and Angelo Bianchi
Bonomi Hemophilia and Thrombosis Center, Fondazione IRCCS Ca’ Granda, Ospedale Maggiore Policlinico, Milan (E.S.) — both in
Italy; Universitätsklinik für Kinder- und Jugendheilkunde, Graz, Austria (W.M.); Hämophiliezentrum, Wabern and Children’s Hospital
of the University of Bern, Bern, Switzerland (R.K.); Division of Hematology/Oncology, Hôpital St. Justine, Montreal (G.R.); and Royal
Hospital for Sick Children, Edinburgh (A.T.).

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Copyright © 2013 Massachusetts Medical Society. All rights reserved.
 Development of Factor VIII Inhibitors in Hemophilia A

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