International Journal of Laboratory Hematology

The Official journal of the International Society for Laboratory Hematology

REVIEW INTERNAT IONAL JOURNAL OF LABORATO RY HEMATO LOGY

Pearls and pitfalls in factor inhibitor assays

D. M. ADCOCK*, E. J. FAVALORO †

*Colorado Coagulation, S U M M A RY
Laboratory Corporation of
Americaâ Holdings, Englewood, The proper performance and interpretation of factor inhibitor
CO, USA assays is a critical role for the hemostasis laboratory. Both false-
†
Department of Haematology,
Institute of Clinical Pathology positive and false-negative inhibitor assays may be reported, lead-
and Medical Research, ing to serious patient mismanagement. Knowledge and recognition
Pathology West, NSW Health of common causes of both false-positive and negative-results can
Pathology, Westmead Hospital,
aid in the identification of these potential pitfalls. Safeguards to
Sydney, NSW, Australia
reporting accurate factor inhibitor assays include initial character-
Correspondence: ization of the sample, using the Nijmegen modification, properly
Dorothy M. Adcock, Colorado performing and interpreting an incubated mixing test in conjunc-
Coagulation, Laboratory
tion, and performing two dilutions for each dependent dilution in
Corporation of Americaâ Hold-
ings, 8490 Upland Dr., Engle- the factor inhibitor assay.
wood, CO 80112-7116, USA.
Tel.: 720 568 4328;
Fax: 720 568 4324;
E-mail: adcockd@labcorp.com

doi:10.1111/ijlh.12352

Received 2 January 2015;

accepted for publication 10
March 2015

Keywords
Factor inhibitor assay, false
positive, false negative, factor
VIII inhibitor, lupus
anticoagulant, EDTA

tion of coagulation test(s), although not all antibodies

INTRODUCTION
Detection and proper characterization of factor inhibi-
tors is essential to patient management yet remains a
challenge for most diagnostic laboratories [1]. Factor
inhibitors are polyclonal immunoglobulins that bind
and neutralize the activity of a coagulation factor
resulting in deficiency of factor activity and prolonga-
are functionally neutralizing [2]. The specific coagula-
tion test(s) prolonged by functional inhibitors depends
on the factor(s) inhibited and its location within the
coagulation cascade. Factor inhibitors may develop in
patients with congenital factor deficiency in response
to factor replacement therapy; those against factor VIII
(FVIII) and factor IX (FIX) comprise the vast majority

52 © 2015 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2015, 37 (Suppl. 1), 52–60

D. M. ADCOCK AND E. J. FAVALORO | FACTOR INHIBITOR ASSAYS
of these alloantibodies. Early detection is crucial to
optimized patient care; thus, surveillance for inhibitor
development in patients under factor replacement
treatment is regularly undertaken [3]. Factor inhibi-
tors may also develop as an autoimmune phenome-
non in individuals without congenital factor
deficiency [4, 5]. The most frequent autologous inhib-
itor is directed against FVIII, and this may lead to a
spontaneous, life-threatening bleeding diathesis [6].
Development of inhibitors in either circumstance
necessitates specific therapy in a timely fashion.
Patient management can be clinically difficult, as they
may not respond to conventional therapies. The
hemostasis laboratory plays a key role in accurate
identification and quantitation of factor inhibitors.
The laboratory assay used to measure factor inhibi-
tors was standardized at a 1975 meeting in Bethesda,
Maryland, and hence is called a Bethesda Assay (BA)
[7]. Modifications of the original assay, referred to as
the Nijmegen–Bethesda assay (NBA), have been intro-
duced to enhance standardization and decrease the
incidence of false-positive results [8]. However, signif-
icant numbers of false-positive (up to 32%) and false-
negative (up to 5%) results are reported, evidenced
by various external proficiency surveys, with little
improvement demonstrated over the past decade [1,
9–14]. This most likely reflects the variety of inhibitor
assay methods used by different laboratories, lack of
incorporation of the Nijmegen modification, poor
appreciation of potential assay interferences and their
impact, as well as incomplete characterization of sam-
ples prior to inhibitor testing. False identification of
the presence or absence of a factor inhibitor may have
tremendous impact on patient care. This article begins
with a review of the incubated mixing assay as its
proper performance and interpretation is integral to
the accurate laboratory evaluation of factor inhibitors.
This is followed by a review of the BA methodology
including the Nijmegen modification. We then focus
on the potential causes of false-positive and false-neg-
ative factor inhibitor assays, concluding with recom-
mendations on how to avoid such misclassifications.
I N C U BAT E D N O R M A L P L A S M A M I X I N G T E S T
These are often performed and interpreted incorrectly
and under inappropriate clinical conditions. Incubated
mixing studies, in which a plasma sample is held at
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53
37 °C for 60–120 min, are indicated in the evaluation
of a prolonged activated partial thromboplastin time
(APTT) when a FVIII inhibitor is in question, such as
in a patient with hemophilia A (i.e. congenital FVIII
deficiency) or a patient with a recent bleeding diathe-
sis. See Figure 1 for proper performance of an incu-
bated mixing test [15]. A time- and temperature-
dependent inhibitor is present when the results of the
patient/normal pool plasma (NPP) mixture are longer
than the control by a given number of seconds or per-
centage, such as, 20%. Prolongation with incubation
is characteristic of FVIII inhibitors and does not occur
with other specific or nonspecific coagulation factor
inhibitors, with the possible exception of FV inhibitors
[16]. A false-positive incubated mixing test may occur
when EDTA plasma is mistakenly used in place of
sodium citrate plasma [12]. While it is reported that
up to 15% of lupus anticoagulants (LA) are time- and
Incubated mixing test
Incubate each at
37° C for 60 to
120 min, then
50% 50% mix paent with
NPP
Paent NPP
Paent + NPP
Compare incubated
mix to control
Separately incubated Paent + NPP
paent + NPP mix Incubated together (Incubated mix)
(Control)
Figure 1. In the proper performance of an incubated
mixing test, three plasma aliquots are prepared. The
first aliquot is patient sample, the second aliquot is
NPP, and the third is patient sample mixed with an
equal volume of NPP. All three aliquots are held at
37 °C for 60–120 min in a water bath. Following
incubation, the separately incubated patient sample
and NPP are combined in equal volume (1 : 1) and
an activated partial thromboplastin time performed
on this mixture (which represents the control), as
well as the patient/NPP mixture that was incubated
(which represents the test). The time in seconds of
the control is compared to the incubated patient/NPP
mixture. A time- and temperature-dependent
inhibitor is present when the results of the patient/
NPP mixture are longer than the control by a given
number of seconds or percentage, such as, 20%.
NPP, normal pool plasma.

54 D. M. ADCOCK AND E. J. FAVALORO | FACTOR INHIBITOR ASSAYS
temperature-dependent, this likely represents an
artifact due to change in pH with incubation, rather
than a characteristic of the inhibitor [17].
BA M E T H O D O L O G Y I N C L U D I N G T H E
N I J M E G E N M O D I F I C AT I O N
Adaptations of BA methodology can be used to identify
not only FVIII inhibitors, but also inhibitors to other
coagulation factors including FXI, FX, FIX, FVII, FV,
and FII, as well as FXIII, protein S and von Willebrand
factor activity [18]. In the original BA assay procedure,
a mixture of patient plasma and NPP incubated at
37 °C for 2 h is compared to a control plasma (created
by combining NPP with an equal volume of buffer) that
is subject to the same assay conditions [7]. Samples
must remain capped whenever feasible to help main-
tain pH [19]. Factor activity of the patient mix and con-
trol plasma is measured following the incubation. For
detection of inhibitors other than FVIII inhibitors, a
10 min- to 1-h incubation at 37 °C is generally consid-
ered sufficient [20]. The residual percent factor activity
is determined and expressed as a ratio of the patient
mix result over the control (see Figure 2). A Bethesda
unit (BU) is defined as the amount of inhibitor that will
inactivate half of the factor present (i.e. results in 50%
residual factor activity) in an equal mixture of patient
and NPP following incubation at 37 °C for 2 h [7].
If an inhibitor is present, inhibitor titer can be deter-
mined using a graph relating inhibitor units to residual
Bethesda assay methodology
Paent mix Control mix
Make diluons of paent plasma
(“dependent diluons”) 1:1 Mix buffer and NPP
1:1 Mix diluons of paent and NPP
Incubate each mix 1–2 h 37 °C water bath
Measure factor acvity at 1:10 and 1:20 diluons
% Residual acvity (RA) = (Paent mix ÷ Control mix) X 100
Bethesda Unit (BU) = 2–log %RA ÷ 0.30; correct for diluons if needed
Nijmegen modificaon uses buffered normal plasma in the paent mix
and FVIIIDP* or 4% albumin rather than buffer in the conrol mix
FVIIIDP: Factor VIII Depleted Plasma
Figure 2. Methodology of the Bethesda assay
including the Nijmegen modification. *FVIIIDP,
factor VIII depleted plasma. NPP, normal pool
plasma.
© 2015 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2015, 37 (Suppl. 1), 52–60
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factor activity or calculated based on the following
formula: BT = (2 log %RA)0.30; being certain to
correct for dilutions if needed, where BT stands for
Bethesda titer and RA (residual activity) [21]. A BT
should be calculated only when the RA falls between
25% and 75%. A RA of 75% correlates to a BT of
0.4 BU, and therefore, this is the lowest possible BT
that can be reported using this method. When the RA is
>75%, the BT is reported as negative. If the RA falls
below 25%, additional dilutions of the patient plasma
should be tested. When multiple residual activities fall
between 25% and 75%, the least dilute sample closest
to 50% is used to determine the BT.
The BA is often performed using multiple dilutions
of patient plasma in buffer so that a BT can be deter-
mined. Patient plasma is typically tested neat, at 1 : 2
and 1 : 5 dilutions, although additional dilutions may
be required with high-titer inhibitors. These initial
patient dilutions are referred to as ‘dependent dilu-
tions’. When the factor activity is performed on these
dependent dilutions following incubation, the analyzer
should be programmed to perform the activity assay at
1 : 10 and 1 : 20 dilutions and these results evaluated
for nonparallelism (Table 1). This step will aid in the
detection of nonspecific inhibitor effect, which could
potentially result in a false-positive BT (Tables 2 and
3). Some nonspecific inhibitors that cannot be distin-
guished in a factor activity assay may be detected in the
dilutions of the BA. This enhanced sensitivity to non-
specific inhibitors in the BA over a factor activity assay
may be due to the added NPP and 37 °C incubation. As
an example, when a strong LA sample without a FVIII
inhibitor is tested in a FVIII activity assay, it may result
in no measurable FVIII activity at all dilutions tested. A
FVIII BA performed on this same sample would appear
positive; however, the 1 : 10 and 1 : 20 dilutions of
dependent dilutions typically show nonparallelism.
The International Society on Thrombosis and
Hemostasis (ISTH) advocates that all laboratories use
the NBA over than traditional BA [22]. The Nijmegen
modification introduces the following two variations:
(i) the use of FVIII-deficient plasma (or 4% BSA)
rather than buffer in the control sample and (ii) buf-
fering of NPP as used in both the patient and control
mixtures [8, 23]. A lyophilized NPP can be reconsti-
tuted with imidazole buffer to create a buffered NPP.
External quality Control of Assays and Tests (ECAT)
and Royal College of Pathologists of Australasia
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D. M. ADCOCK AND E. J. FAVALORO | FACTOR INHIBITOR ASSAYS 55

Table 1. Example of a Bethesda assay calculation sheet with a high-titer factor VIII (FVIII) inhibitor

FVIII Bethesda assay calculation sheet

Patient tube FVIII

activity
Dependent Control Log residual BU 9 dilution
Patient dilution 1 : 10 1 : 20 Av factor activity %RA activity BU factor

Smith 1 0 0 0 45 0 ncalc ncalc ncalc

20 0 0 0 45 0 ncalc ncalc ncalc
40 5.2 0 3 45 7 0.82 3.9 156
80 8.1 9.9 9 45 20 1.30 2.3 186
160 11.7 12.7 12 45 27 1.43 1.9 305
320 16.0 16.3 16 45 36 1.55 1.5 477
640 23.1 25.2 24 45 53 1.73 0.9 580
1280 33.3 32.0 33 45 73 1.87 0.45 573

RA, residual activity; BU, Bethesda unit; ncalc, unable to calculate; av, average.
Example of a factor VIII inhibitor calculation sheet using the following formula: BT = (2 log %RA)/0.30 and correct-
ing for the dependent dilutions. This factor VIII inhibitor has a titer of 580 BU. Italics indicated the value of the titer.

Table 2. Example of a factor IX Bethesda assay calculation sheet with a high-titer factor VIII inhibitor without
performing two dilutions for each dependent dilution resulting in a false-positive factor IX (FIX) inhibitor titer

FIX Bethesda assay calculation sheet

Patient tube Control factor Log residual BU 9 dilution

Patient Dilution FIX activity activity %RA activity BU factor

Jones 1 0 53 0 ncalc ncalc ncalc

2 0 53 0 ncalc ncalc ncalc
5 9.3 53 18 1.24 2.51 12.6
10 15.8 53 30 1.47 1.75 17.5
20 24.4 53 46 1.66 1.12 22.4
40 37.2 53 70 1.85 0.51 20.4
80 42.5 53 80 1.90 0.32 25.5
160 50.4 53 95 1.98 0.07 11.6

ncalc, unable to calculate; RA, residual activity; BU, Bethesda unit.

Example of a factor IX inhibitor calculation sheet using the following formula: BT = (2 log %RA)/0.30 and correct-
ing for the dependent dilutions. This strong factor VIII inhibitor gives a factitious FIX inhibitor titer of 22.4 BU. Italics
indicated the value of the titer.

(RCPA) surveys indicate that only about 30% of par-
ticipants use the Nijmegen modification [10, 14].
P OT E N T I A L C AU S E S O F FA L S E - P O S I T I V E BA S
Lupus anticoagulants
Lupus anticoagulants are a heterogeneous population
of antibodies directed against phospholipid-binding
proteins. These nonspecific inhibitors of coagulation
may interfere with APTT-based factor activity assays
(i.e. FVIII, FIX, FXI, and FXII) causing one or more
spuriously low or undetectable factor activity results
[10, 24, 25]. Lupus anticoagulants inhibitor effect in
factor assays can often be diluted with saline or buffer
leading to increasing factor activity at the 1 : 10,
1 : 20, and 1 : 40 dilutions (nonparallelism) [26]. Par-
ticularly strong LA, however, may not dilute out

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56 D. M. ADCOCK AND E. J. FAVALORO | FACTOR INHIBITOR ASSAYS

Table 3. Example of a factor IX (FIX) Bethesda assay calculation sheet with a high-titer factor VIII (FVIII) inhibitor
demonstrating nonparallelism in the two dilutions for each dependent dilution suggesting further investigation
before reporting

FIX Bethesda assay calculation sheet

Patient tube FIX

activity
Control factor Log residual BU 9 dilution
Patient Dilution 1 : 10 1 : 20 Av activity %RA activity BU factor

Jones 1 0 0 0 53 ncalc ncalc ncalc ncalc

2 0 0 0 53 ncalc ncalc ncalc ncalc
5 9.3 17.0 * 53 ncalc ncalc ncalc ncalc
10 15.8 27.3 * 53 ncalc ncalc ncalc ncalc
20 24.4 34.0 * 53 ncalc ncalc ncalc ncalc
40 37.2 48.8 * 53 ncalc ncalc ncalc ncalc
80 42.5 49.3 * 53 ncalc ncalc ncalc ncalc
160 50.4 55.1 * 53 ncalc ncalc ncalc ncalc

*Nonparallelism evident, further investigation needed to rule out nonspecific inhibitor effect; ncalc, unable to calculate;
RA, residual activity; BU, Bethesda unit.
Example of a factor IX inhibitor calculation sheet using the following formula: BT = (2 log %RA)0.30 and correct-
ing for the dependent dilutions. This strong factor VIII inhibitor demonstrates nonparallelism in the 1 : 10 and 1 : 20
dilutions of each FIX activity-dependent dilution indicating a nonspecific inhibitor effect which reflects the FVIII inhibi-
tor neutralizing the FVIII in the FIX-deficient plasma used in the assay.

resulting in one or more intrinsic factor activity assays
to appear low or even undetectable. This interference
can also produce false-positive BA results [9]. The lab-
oratory distinction between an LA and specific intrin-
sic factor inhibitor can be difficult [10, 25]. In
proficiency surveys conducted by the National Exter-
nal Quality Assessment Service (NEQAS), LA positive
(and FVIII inhibitor negative) samples were incor-
rectly classified as a FVIII inhibitor by 6.5% of partici-
pants in 1998 and 4.4% in 2007 [13]. Clinically, this
is a critically important distinction and such misinter-
pretation can significantly influence patient manage-
ment. Patients with LA are potentially at an increased
risk for thrombosis, while factor inhibitors generally
lead to a bleeding diathesis.
One clue to LA interference in the BA is the dem-
onstration of nonparallelism in the 1 : 10 and 1 : 20
dilution results of the dependent dilutions as evi-
denced by increasing activity with increasing dilution.
Another clue is that multiple intrinsic factor activities
may appear to be reduced and multiple intrinsic fac-
tors may have positive BT. It is also useful to pay
attention to the results of the incubated mixing study
as FVIIII inhibitors typically demonstrate time- and
temperature-dependent APTT prolongation, while LA
tend to act as immediate inhibitors. [25]. High-titer
FVIII inhibitors, however, can be ‘fast acting’ (or high
levels capable of swamping the available FVIII) and
demonstrating inhibition with immediate NPP mix
with little if any further prolongation with incubation.
Further complicating the issue is that FVIII inhibitors
may yield false-positive results in some LA assays, spe-
cifically the hexagonal phospholipid assay. Dilute Rus-
sell viper venom (dRVVT)-based LA assays are not
interfered by FVIII inhibitors, and thus, a positive LA
test result by dRVVT testing can be used with some con-
fidence to distinguish LA and FVIII inhibitors. Patients
with FVIII inhibitors can nevertheless have coincident
LA [27]. In the distinction between true FVIII inhibitors
and spurious positive results due to LA, chromogenic
FVIII activity and ELISA-based FVIII inhibitor assays
are extremely useful as neither assay demonstrates LA
interference. Naturally, additional approaches and clues
can help distinguish LA and specific factor inhibitors,
with clinical presentation being one key ingredient –
patients with LA are predisposed to thrombosis, while
those with specific factor inhibitors would classically
present with a bleeding diathesis.

© 2015 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2015, 37 (Suppl. 1), 52–60

D. M. ADCOCK AND E. J. FAVALORO | FACTOR INHIBITOR ASSAYS
Specific factor inhibitor present but is other than the
factor inhibitor under investigation
High-titer factor inhibitors may interfere with factor
activity assays other than the factor for which the inhib-
itor is directed [28]. For example, a high-titer FVIII
inhibitor may inhibit the FVIII present in the normal
plasma used in a FIX activity assay, prolonging the
APTT, leading to a spuriously low FIX activity and posi-
tive FIX BA. High-titer FVIII inhibitors occasionally may
not be able to be diluted out and may result in very low
or even undetectable FIX, FXI, or FXII activity at the
1 : 10, 1 : 20, and 1 : 40 dilutions. This may also result
in false-positive FIX, XI, and/or XII BAs. If the labora-
tory is requested to perform, for example, only a FIX
BA on this FVIII inhibitor-containing sample, they may
report out a false-positive FIX inhibitor result (see
Tables 2 and 3). A similar situation may also occur
when laboratories encounter more unusual factor
inhibitors (e.g. to FV, FX, or FXI). When testing samples
with these more unusual inhibitors, a laboratory may
recognize the presence of an inhibitor, but have a
request to test only for the more common inhibitors,
such as FVIII or FIX and therefore the potential to
report either of these as positive. This has a greater like-
lihood of occurrence when more common inhibitors are
tested in a local laboratory, and more esoteric inhibitor
assays are sent to a reference laboratory. A 2005 RCPA
QAP survey distributed a strong factor V inhibitor sam-
ple (complicated by a coincident LA) to 42 laboratories.
Only 63.4% of participants identified the factor V inhib-
itor, 4.8% identified a FVIII inhibitor, and 17.1% identi-
fied LA only [11]. If, for example, a specific FVIII
inhibitor was subject to a FIX BA, the 1 : 10 and 1 : 20
dilution results of the dependent dilutions typically
demonstrate nonparallelism. Another clue would be
that multiple intrinsic factors (i.e. FVIII, FIX, FXI, FXII),
if tested, may have positive BTs, with the highest titer
pointing to the true specific factor inhibitor. For exam-
ple, a high-titer FVIII inhibitor may yield a FVIII BT of
1200 BU, but FIX and FXI BAs performed on the same
sample would have titers in the range of 1–20 BU. Eval-
uation of the results of multiple factor assays may pro-
vide another clue pointing to the true specific factor
inhibitor. For example, a FVIII inhibitor is likely if the
FVIII activity is undetectable, but FIX, FXI, and FXII
activities are each low but measurable. Attention to the
results of the incubated mixing test may also be useful
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57
as only FVIII inhibitors demonstrate time and tempera-
ture dependence.
EDTA plasma
Sodium citrate is the sample matrix required for fac-
tor inhibitor assays. Samples collected in potassium
EDTA must result in sample rejection. While it is easy
to identify the incorrect matrix when samples are
submitted as primary tubes, EDTA plasma, serum,
and citrate plasma appear identical in secondary
aliquots.
EDTA is a very strong calcium chelator resulting in
spuriously low FVIII and FV activity results and mod-
erate prolongation of both the APTT and PT [19].
Incubated mixing studies using EDTA plasma result in
time- and temperature-dependent prolongation in
both APTT and PT mixing tests [12]. Furthermore,
EDTA plasma may yield false-positive FVIII and FV
BTs, typically in the range of 1–5 BU. In a 2005 RCPA
QAP, 68.3% of 42 laboratories falsely identified a FV
and/or FVIII inhibitor in normal EDTA plasma [11].
If a FV or FVIII BA is performed in EDTA plasma,
dependent dilutions run at 1 : 10 and 1 : 20 dilutions
do not demonstrate nonparallelism. As EDTA plasma
demonstrates prolongation of the APTT, prolongation
with an incubated mixing test, decreased FVIII activity
and a positive BA, EDTA plasma can perfectly mimic
a FVIII inhibitor.
Clues to the presence of a false-positive FVIII
inhibitor secondary to an otherwise normal EDTA
plasma include a clinically unexpected result; for
example, a positive FVIII inhibitor in a pediatric
female patient with no history of bleeding. Another
clue is that EDTA plasma results in prolongation of
both the APTT and PT. FVIII inhibitors prolong the
APTT only, although factor V inhibitors elevate both
the APTT and PT. A chromogenic FVIII activity is fal-
sely low when performed with EDTA plasma. An
ELISA-based FVIII inhibitor assay would be negative
with otherwise normal EDTA plasma. To confirm the
sample is EDTA rather than citrated plasma, standard
chemistry parameters including potassium, sodium,
and calcium can be performed. EDTA plasma yields
absent calcium, elevated potassium, and normal
sodium levels, while sodium citrate plasma yields ele-
vated sodium, detectable calcium, and normal potas-
sium levels [29].

58 D. M. ADCOCK AND E. J. FAVALORO | FACTOR INHIBITOR ASSAYS
Anticoagulant therapy
Novel oral anticoagulants including both direct
thrombin and FXa inhibitors are being increasingly
used for long-term anticoagulation. These anticoagu-
lants, as well as heparin, may cause spuriously
decreased factor activity results and falsely positive
BAs, depending on anticoagulant concentration and
responsiveness of the APTT or PT reagent to these
agents [30–33]. Activated partial thromboplastin time
and PT reagents vary significantly in their respon-
siveness to each of these anticoagulant drugs [32–
34]. For this reason, the potential impact of these
agents on factor activity and inhibitor assays depends
on the specific drug, its plasma concentration, and
the APTT and/or PT reagent in use. It is inappropri-
ate to perform a factor inhibitor assay in the
presence of a direct IIa or Xa inhibitor anticoagulant
or heparin that has not been neutralized in the
laboratory.
In the emergency management of a bleeding
patient taking a direct thrombin or Xa inhibitor anti-
coagulant, the patient may be mistakenly diagnosed
as suffering with a factor inhibitor. This is especially
likely if medication history is unavailable or the treat-
ing physician does not understand the effect of these
anticoagulant drugs on coagulation assays. Heparin,
direct thrombin, and direct Xa inhibitors at significant
concentration may lead to elevation of the APTT with
lack of correction in a 1 : 1 plasma mix [16]. Intrinsic
factor activities may be falsely low, although nonpar-
allelism can often, but not consistently, be demon-
strated [30, 31]. A BA yields a positive inhibitor titer
although the 1 : 10 and 1 : 20 dilutions of the depen-
dent dilutions should demonstrate nonparallelism
[30–32].
Clues to the presence of an anticoagulant drug lie
in the results of screening assays. More information is
available in Assessing Nonvitamin K Antagonist Oral Anti-
coagulants (NOACs) in the Laboratory in this issue.
Laboratory error/analytical issue
False-positive factor inhibitor identification in normal
plasma and/or serum as well as in factor-deficient
plasma has been reported and occurs more readily
when a standard BA rather than Nijmegen-modified
assay is used. External quality Control of Assays and
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Tests reported false identification of an inhibitor by
8–17% of participants when testing FVIII-deficient
plasma [10]. A false-positive FVIII inhibitor can be
reported in a patient with a high-level FV inhibitor, as
described earlier.
P OT E N T I A L C AU S E S O F FA L S E LY N E G AT I V E
N I J M E G E N – B E T H E S DA T I T E R R E S U LT S
Recent treatment with factor or by-passing agent
In factor-deficient patients receiving replacement ther-
apy, inhibitor testing is recommended when there is
poor clinical response to treatment or if post-therapy
factor levels are lower than expected. Inhibitor tests
are most sensitive after the patient’s factor level has
returned to baseline for 24 h. Residual factor may
mask or extinguish a low-titer inhibitor. If residual
concentrate is present in the patient sample, inactiva-
tion of residual factor, for example, by heating the
plasma to 58 °C for 90 min, is recommended. Resid-
ual factor antigen following heat inactivation, how-
ever, may still interfere with the BA, yielding a lower
BT than if the assay were performed in the absence of
residual heat-inactivated factor.
Laboratory error/analytical issue
EQA data also report the failure of laboratories to rec-
ognize the presence of an inhibitor when one is pres-
ent. This is most likely to occur when low-level
inhibitors of approximately 1.0 BU/mL are present
and if a classic BA is used rather than the Nijmegen
modification [1, 14]. Interestingly, differential buffer-
ing used by different laboratories (e.g. in house vs.
commercial buffered normal plasma) will also differ-
entially affect whether or not laboratories correctly
identify these low-titer inhibitors [1, 18]. Laboratories
should be encouraged to evaluate their own assay’s
low-level sensitivity.
S A F E G UA R D S TO R E P O RT I N G FA L S E -
P O S I T I V E O R FA L S E - N E G AT I V E BA S
There are a number of steps laboratories can take to
help safeguard against reporting falsely positive or
negative factor inhibitor assays. One is to characterize
all samples by measuring an APTT, PT, and thrombin
 1751553x, 2015, S1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ijlh.12352 by Cochrane Saudi Arabia, Wiley Online Library on [16/06/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
D. M. ADCOCK AND E. J. FAVALORO | FACTOR INHIBITOR ASSAYS 59

clotting time prior to performing inhibitor assays. This
is especially useful in reference laboratories that
receive secondary aliquot samples often without inclu-
ded history. Sample characterization will also help in
identifying serum, EDTA plasma, or anticoagulant
therapies, all of which may cause a false-positive
inhibitor result. The presence of direct Xa inhibitor
anticoagulants is more difficult to identify than hep-
arin or direct thrombin inhibitor anticoagulants using
routine clotting assays, as some agents, particularly
apixaban, may have little effect on the APTT and PT.
A chromogenic anti-Xa assay can detect the presence
of direct Xa inhibitor agents. Laboratories without
the capability to run anti-Xa assays may need to rely
on the patient’s medication history.
Proper performance and interpretation of incu-
bated mixing tests and correlation to inhibitor assays
results is important. An incubated mixing test is
indicated when a FVIII inhibitor is under investiga-
tion. If a sample with a prolonged APTT demon-
strates correction or near correction on addition of
normal plasma, but no further prolongation with
incubation, a FVIII inhibitor is likely not present.
Likewise, a sample that demonstrates time- and
temperature-dependent prolongation warrants mea-
suring FVIII activity if not already ordered. Impor-
tant caveats include EDTA plasma, novel oral
anticoagulants, and some LA, which can mimic FVIII
inhibitors. A chromogenic FVIII activity does not
show LA interference and can be useful, as can be a
dRVVT to help determine LA.
Implementation of the Nijmegen modification of the
BA is recommended to avoid reporting false-positive
and false-negative inhibitor results. In addition, it is
useful to measure baseline factor activity prior to the
BA to determine whether heat inactivation is needed. If
the baseline activity is too high, a BA is not indicated.
A final safeguard in performing the BA is to
include 1 : 10 and 1 : 20 dilutions in the factor activ-
ity assay performed on the dependent dilutions. Non-
parallelism suggests a nonspecific inhibitor and the
need to further investigate the sample.
CONFLICT OF INTEREST
The authors declare no conflict of interest.

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