283

Mixing Tests: Diagnostic Aides in the

Investigation of Prolonged Prothrombin Times
and Activated Partial Thromboplastin Times
Geoffrey Kershaw, FAIMS1 Daniel Orellana, BSc1

1 Haemostasis Laboratory, Institute of Haematology, Royal Prince Address for correspondence Geoffrey Kershaw, FAIMS, Institute of
Alfred Hospital, Camperdown, New South Wales, Australia Haematology, Royal Prince Alfred Hospital, Missenden Road,
Camperdown, New South Wales 2065, Australia
Semin Thromb Hemost 2013;39:283–290. (e-mail: Geoffrey.Kershaw@sswahs.nsw.gov.au).

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Abstract Mixing tests are a relatively simple procedure used in the hemostasis laboratory as a first-
line investigation into the cause of an abnormal screening test, typically a prolonged
activated partial thromboplastin time and/or a prolonged prothrombin time. The
mixing test involves combining the test plasma with normal plasma, then repeating
the screening test on the mixture to assess whether the clotting time becomes normal
or remains prolonged. The primary purpose of a mixing test is to guide further
investigations. When mixing test results “normalize,” this suggests the test plasma is
deficient in clotting factor(s) and thus specific factor assays can be performed to
determine which are reduced. When the mixing test result does not “normalize,” this
suggests the presence of an inhibitor or other type of interference (e.g., the presence of
an anticoagulant such as high-dose heparinoids), and so the laboratory needs to
determine if this is a lupus anticoagulant or a specific coagulation factor inhibitor, or
another type of inhibitor. Because these follow-up investigations are more costly and
time-consuming than the basic screening tests, the appropriate performance and
interpretation of mixing tests is advantageous for the laboratory. Moreover, the correct
laboratory approach is also clinically relevant, as patient management is ultimately
affected, and an incorrect interpretation may lead to inappropriate therapies being
established. Components of a mixing test that can influence result interpretation
include the sensitivity of the used screening reagents to various factor deficiencies and
Keywords inhibitors, the source or composition of the normal plasma, and the setting of cutoffs for
► PT the formula used in expressing mixing test results. Numerous and differing criteria for
► APTT mixing test interpretation have been suggested historically, which can lead to confusion
► mixing test as to which approach is the most appropriate. The use of differing criteria will also lead to
► factor deficiency differing interpretations regarding “normalization.” For this pivotal reason, standard-
► inhibitors ized mixing test procedures and a consistent set of validated interpretive criteria
► correction represent the most favorable approach to maximizing the utility of a mixing test, and
► non-correction ensure the most accurate diagnosis for investigated patients.

The mixing test is a routinely performed assay in the hemo- acquired hemophilia)2,3 and antiphospholipid antibodies
stasis laboratory, and it is an essential test in the diagnostic syndrome,4 as well as for troubleshooting other less frequent
approach of clotting factor deficiencies,1 other severe disor- pathological or physiological conditions.5 The reason for
ders such as acquired inhibitors of coagulation factors (i.e., performing a mixing test entails typically a prolongation of

published online Issue Theme Quality in Hemostasis and
March 2, 2013 Thrombosis, Part II; Guest Editors,
Giuseppe Lippi, MD, Mario Plebani, MD,
and Emmanuel J. Favaloro, PhD,
FFSc (RCPA).
Copyright © 2013 by Thieme Medical DOI http://dx.doi.org/
Publishers, Inc., 333 Seventh Avenue, 10.1055/s-0033-1336832.
New York, NY 10001, USA. ISSN 0094-6176.
Tel: +1(212) 584-4662.
284 Mixing Tests in the Investigation of Prolonged PT and APTT Kershaw, Orellana

one of or both prothrombin time (PT) and activated partial
thromboplastin time (APTT). The combination of the PT and
APTT provides the most commonly applied general screen for
deficiencies of the coagulation factors, comprising tissue
factor and contact factor pathways of the hemostatic system,
respectively (►Fig. 1). The longer pathway is the contact
factor (or “intrinsic”) pathway, which is started in vitro by
the reciprocal activation of the contact factors prekallikrein
(PK, Fletcher factor) and FXII in the presence of high-molecu-
lar-weight kininogen (HMWK). These contact factors are
needed for initiation of the coagulation cascade as reflected
in vitro by the APTT, and also by the silica clotting time (CT)
test and kaolin CT test. The tissue factor (or “extrinsic”)
pathway begins with the binding of tissue factor with FVII
prolonged CT must be made before proceeding to a mixing
test, as preanalytic errors are still the leading source of
uncertainty in laboratory testing7 (►Fig. 2). Preanalytic phase
errors that affect PT and APTT include incorrect anticoagulant
type (e.g., heparin or EDTA contamination), under-filled
tubes, hemolysis, activated or clotted samples, and incorrect
labeling, and each of these can compromise result quality.8–11
The mixing test chosen should be based on the same test
principle used to detect the prolonged CT. For example, if the
PT is prolonged and the APTT normal, then only a PT mixing
test is required. If both PT and APTT are prolonged, then two
mixing tests can be performed. Although the principle of the
mixing test is simple, a large number of patient-related and
laboratory-related variables will influence the outcome of

to form a complex that activates FX, leading to the first part of
the “common” pathway of the cascade.6
The CT is the time taken (in seconds) from addition of the
starting reagents to the formation of an insoluble fibrin clot. A
CT above the upper limit of the laboratory’s reference interval
can be caused either by a deficiency of one or more of the
clotting factors contained in the coagulation pathways or by
the presence of anticoagulants (e.g., heparinoids) or inhib-
itors that interfere with the cascade, even in the presence of
normal levels of clotting factors. A mixing test might be
warranted when a prolonged CT is observed in the absence
of anticoagulant therapy or other identifiable causes, such as
liver disease or preexisting hemophilia. Efforts to check for
and exclude preanalytic phase errors as a cause of the
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mixing tests. These include the specific factor(s) that may be
deficient and the severity of the deficiency; the nature and
strength of the inhibitor that may be present; the choice of
normal pooled plasma (NPP) for the mixing test; the factor-
deficiency sensitivity or inhibitor sensitivity of PT and APTT
reagents; and the method of result interpretation. Although
the majority of mixing tests are probably performed in a 1:1
ratio of test plasma to NPP, variations exist such as 4:1 and 1:4
ratios that, in some instances, may clarify interpretations.
Mixing Tests and Factor Deficiencies
The various types of factor deficiencies and inhibitors that
may be encountered in the hemostasis laboratory are

Fig. 1 Diagrammatic representation of the two major clotting pathways (prothrombin times [PT] and activated partial thromboplastin times
[APTT]). The names in italics are the names of some other clotting time tests and the points at which they act in the cascade. The APTT test is
potentially sensitive to prolongation when there is a deficiency of any clotting factor except FVII. The PT is sensitive to loss of FVII but insensitive to
loss of factors above FX in the contact factor pathway. When a test plasma has a prolonged PT and/or APTT that corrects in a mixing, performing
additional tests such as the Russell’s viper venom (RVV) time, Echis time, thrombin time, and reptilase time can assist in locating which region of
the cascade the deficiency is located.

Seminars in Thrombosis & Hemostasis Vol. 39 No. 3/2013

 Mixing Tests in the Investigation of Prolonged PT and APTT
Fig. 2 Flowchart for the investigation of prolonged screening tests.
Before performing a mixing test, it is useful, where possible, to exclude
preanalytic variables such as an under-filled citrate collection tube or
the presence of a fibrin clot. The thrombin time test, when prolonged
in conjunction with a prolonged activated partial thromboplastin
times (APTT), may point to the presence of heparin in plasma as a
contaminant. When the mixing test shows a correction, specific factor
assays can be performed to identify the reduced factor(s). If the mixing
test shows non-correction, then assays for the lupus anticoagulant or
specific factor inhibitor can be performed. Knowledge of the patient’s
clinical history may be useful in directing which inhibitor to test for, as
lupus anticoagulant predisposes to a thrombosis and factor inhibitors
to a bleeding tendency.
summarized in ►Table 1. Prolongation of CTs above the upper
limit of the laboratory’s reference interval combined with a
mixing test result that “normalizes” is said to “correct” and is
indicative of factor deficiency. There are numerous PT and
APTT reagents available in the market, and each will have its
own factor-deficiency sensitivity profile. This means the level
below which, for example, FVIII must fall before the APTT
becomes prolonged will not be the same for all APTT reagents,
nor is this level likely to coincide with the laboratory’s
reference interval lower limit for that factor. The APTT
reagents should be sensitive to deficiencies of factors VIII,
IX, and XI at concentrations of 35 to 40 U/dL.12
There is wide variability in the relative frequencies of
inherited deficiencies of specific coagulation factors in the
general population and therefore the frequency with which
these are encountered in the laboratory. The X-linked defi-
ciencies of FVIII and FIX, combined with von Willebrand
disease, make up the vast majority of inherited factor defi-
ciencies associated with a bleeding disorder.13 The presum-
ably homozygous forms of the rarer factor deficiencies range
in frequency from 1 in 2 million for FII deficiency to 1 in
500,000 for FVII deficiency.13,14 The combined prevalence of
moderate and severe inherited FXII deficiency has been
estimated at approximately 2.3% of the central European
population.15 Although there is no bleeding phenotype asso-
ciated with FXII deficiency, this may be the most common
Kershaw, Orellana 285
cause of an isolated prolonged APTT that corrects on
mixing.16
When a factor deficiency is detected with the mixing test,
specific factor assays are normally needed to determine
which factor is deficient. When the APTT is prolonged and
the PT is normal, a standard protocol would be to perform
individual assays for FVIII, FIX, FXI, and FXII, as these factors
are those that are unique to the APTT (►Fig. 1). Deficiencies of
PK and HMWK may also prolong the APTT and correct on
mixing, but they are more rarely encountered and are not
generally considered diagnostically important because, like
FXII, they are not associated with a bleeding phenotype.16 The
basis of correction in a CT due to mixing is that a normal CT
will occur with 50% or more of all coagulation factors. Thus,
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even mixing patient plasma devoid of clotting factors with
NPP (representing approximately 100% of each factor by
definition) will correct.
Mixing Tests and Inhibitors
From the hemostasis laboratory’s perspective, inhibitors are
something in the test plasma that prolongs one or more CTs,
such that the CT remains significantly prolonged after the test
plasma is mixed with NPP. This non-normalization or non-
correction upon mixing indicates that the initial prolongation
was not due to a factor deficiency. Inhibitors can be global in
nature, such as the lupus anticoagulant (LA), which prolongs
CTs in the lack of inherited factor(s) deficiency, or they can be
directed against a specific factor, such as FVIII antibodies seen
in congenital or acquired hemophilia A (AHA).
LA tends to preferentially prolong the APTT with the PT
being only rarely prolonged. Even so, there are large differ-
ences in sensitivity to LA among APTT reagents (►Table 2)4
such that one laboratory may detect the inhibitor and per-
form a mixing test followed by specific tests for LA, while the
APTT may be within the reference interval therefore with no
follow-up required in another laboratory using a LA-insensi-
tive APTT reagent. The laboratory needs to be aware of the LA
sensitivity of the reagent in use, as general background
knowledge to assist in mixing test interpretation. If a labora-
tory is using a relative LA-insensitive reagent such as Actin FS
(Siemens, Marburg, Germany), it may then be beneficial to
use a more LA-sensitive reagent to perform the mixing test
when the prolongation of APTT is by just a few seconds.
Testing plasma by two APTT reagents with differing LA and
factor-deficiency sensitivities can give insight to the nature of
the abnormality ahead of the mixing test results.
Occasionally, global inhibitors such as a very strong LA can
cause an apparent reduction in activity of all clotting factors
(especially those of the intrinsic pathway, i.e., FVIII, FIX, FXI,
and FXII) as measured by one-stage assays with a lupus-
sensitive APTT reagent.17,18 It is important to be aware of this
phenomenon, so as to avoid misinterpretation of the LA with
a specific factor inhibitor. This could occur if the patient had a
clinical presentation of bleeding, with the low factor level
assumed to be the result of a factor inhibitor. The LA in this
setting would also interfere with a subsequent Bethesda
assay, thus compounding the misinterpretation.19
Seminars in Thrombosis & Hemostasis Vol. 39 No. 3/2013
286 Mixing Tests in the Investigation of Prolonged PT and APTT Kershaw, Orellana

Table 1 Classification of plasma factor deficiencies and inhibitors and their typical behavior in clotting time tests

Clotting time test

PT APTT PTmix APTTmix TT dRVVT Echis Reptilase
Inherited factor deficiencies
FVIII/FIX/FXI/FXII/PK/HMWK N R cor N N N N
FII R R cor cor N R R N
FV/FX R R cor cor N R N N
FVII R N cor N N N N
Fibrinogen/dysfibrinogenemia R R cor cor R R R R
FXIII N N N N N N
VWF (with low FVIII) N R cor N N N N

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Acquired factor deficiencies
Liver disease R R cor cor N/R R R N/R
Vitamin K deficiency/warfarin ingestion R R cor cor N R N N
Disseminated intravascular coagulation R R cor cor R R R R
Amyloidosis (with loss of FX) R R cor cor N R N N
Nephrotic syndrome (with loss of FXII) N R cor N N N N
Low FVIII secondary to VWF inhibitor N R cor N N N N
Immunoglobulin-type inhibitors
Lupus anticoagulant N R n-c N R N N
FVIII inhibitor N R n-c
N N N N
FIX or FXI inhibitor N R n-c N N N N
FV or FX inhibitor R R n-c n-c N R N N
Therapeutic agents
Unfractionated heparin N R n-c R N R N
Lepirudin R R n-c n-c R R R N
Dabigatran (FIIa inhibitor) R R n-c n-c R R R N
Rivaroxaban (FXa inhibitor) R R n-c n-c N R N N

Abbreviations: APTT, activated partial thromboplastin time; cor, correction; dRVVT, diluted Russell viper venom time; HMWK, high-molecular-weight
kininogen; N, normal; n-c
, slow-acting non-correction; n-c, non-correction; PK, prekallikrein; PT, prothrombin time; R, raised; TT, thrombin time; VWF,
von Willebrand factor.
Note: The presence of and/or degree of raised clotting times in tests can vary with the degree of factor deficiency and reagent sensitivity to particular
factor deficiencies or global inhibitors.

Table 2 Demonstration of the wide variation in sensitivity of APTT reagents to the presence of lupus anticoagulants in plasma

APTT in seconds
TriniCLOT APTT S TriniCLOT APTT HS Actin FSL Actin FS PTT-LA
Upper limit 34 35 40 36 54
Patient 1 62 61 65 31 132
Patient 2 88 74 74 34 138
Patient 3 234 186 164 47 > 300
Patient 4 60 64 69 30 253
Patient 5 154 171 136 66 258

Abbreviation: APTT, activated partial thromboplastin time.

Notes: Samples are from five individuals with lupus anticoagulant demonstrated by the diluted Russell viper venom test. The upper limit (mean þ 2 SD)
determined on 50 normal samples is also shown.

Seminars in Thrombosis & Hemostasis Vol. 39 No. 3/2013

 Mixing Tests in the Investigation of Prolonged PT and APTT Kershaw, Orellana 287

Inhibitors to single coagulation factors occur far less
frequently than LA. The most frequently observed single
factor inhibitor is directed against FVIII. In persons without
a prior bleeding history, this leads to the condition of AHA,
which has an incidence of 1.48 per million persons per year.20
These individuals usually have FVIII inhibitors with different
kinetics when compared with FVIII inhibitors seen in indi-
viduals with congenital hemophilia A.19,21 This means FVIII:C
levels in plasma of up to 10 U/dL or even higher can coexist
even with a very high titer FVIII inhibitor. FVIII inhibitors
show slow acting kinetics, which is often also temperature
dependent, so that mixes require incubation at 37°C for 1 or 2
hours for full expression of their activity. To be classified as a
FVIII inhibitor, there must be an increase between the APTT
cases, the presence of a common pathway factor inhibitor
such as a FV inhibitor can be excluded due to the correcting
PT.24 The non-correcting APTT suggests the presence of an
inhibitor, the nature of which still needs to be elucidated by
further tests including factor assays and LA testing. If an
inhibitor develops in a patient with congenital hemophilia A
or B, the PT will be unchanged (remains normal), but the APTT
mixing test will switch from correcting pattern before the
inhibitor development to non-correcting pattern.
Normal Plasma for Mixing Tests
The NPP used in a mixing test ideally has close to 100 U/dL of
all the clotting factors so that a 1:1 mix with patient plasma

measured immediately upon mixing and the APTT measured
after extended incubation. A minimum difference of 10 sec-
onds has been suggested.22 The difference achieved will be
influenced by amount of FVIII present in the patient’s plasma
and the effect this has on the initial APTT, with some APTTs
being only moderately prolonged. Although FVIII inhibitors
are generally accepted as being slow acting, the neutralization
of FVIII in the NPP begins immediately upon mixing, with a
rise in the APTT being seen in as little as 10 minutes.18 It is
important, therefore, to minimize any delay in performing the
APTT on the immediate mix tube so as to maximize the
chance of observing a difference between the CTs of the
immediate and incubated mixes. One method of achieving
this is to program the coagulation analyzer, if possible, to do
the immediate mix test. The incubation period itself can cause
some loss of FV and FVIII, given that the incubation is
performed at 37°C, and that these are labile coagulation
factors. This effect can be negated by incubating separate
tubes of patient’s plasma and of NPP, alongside the 1:1 mix of
the patient’s plasma and NPP. At the end of the 1 to 2 hours
incubation, the separately incubated patient’s plasma and
NPP are mixed and tested immediately for APTT.23
Occasionally, patient plasma may contain both factor
deficiency and an inhibitor, for instance, LA in the presence
of multiple factor deficiency (as in liver disease) or LA in the
presence of a common pathway factor deficiency. If PT and
APTT mixing tests are performed in this situation, the PT will,
in most cases, correct and the APTT will not correct. In these
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will contain at least 50 U/dL of all factors. This factor level
ensures that a correction is possible in the absence of an
inhibitor. NPP can be prepared in-house from routine normal
samples or sourced commercially as either frozen plasma or
lyophilized plasma. Because the concentration of some clot-
ting factors can vary up to fourfold in different normal plasma
samples, the larger the number of donors in the pool, the
more likely the pool clotting activity will be close to 100 U/dL,
with a minimum of 20 donors suggested.12 Some caution is
needed if “normal plasma” from patients is used, as these
frequently contain high levels of FVIII. The NPP should be
double centrifuged to ensure a platelet count of less than
10  109/L before freezing.25
Setting Cutoff Levels for Mixing Test
Interpretation
Several different approaches to setting cutoff levels for rou-
tine mixing test interpretation have been proposed
(►Table 3), but there appears to be no universally accepted
method. One method is to look at the CT of the 1:1 mix relative
to a fixed value such as the upper limit of the reference
interval for PT or APTT used. A correction occurs with this
approach if the 1:1 mix CT falls below the limit. Other
methods include, but are not limited to, the following: (i)
seeing if the mix CT correction is within the mean normal CT
plus 2 to 3 standard deviations; (ii) expressing the mix CT as a
simple ratio to the NPP CT, with a correction occurring when

Table 3 Examples of cutoff levels for mixing test interpretation

1. Methods that use only the 1:1 mix CT

a. Comparing the 1:1 mix CT with the upper limit of the reference interval
b. Comparing the 1:1 mix CT with the mean normal CT plus 2 SD or plus 3 SD
2. Methods that use the 1:1 mix CT and NPP CT
a. Comparing the ratio of 1:1 mix CT to NPP CT to a level such as 1.1 or 1.2
b. Comparison of the difference in CTs between the 1:1 mix and NPP with a preset difference, such as 5 seconds.
c. Comparing the normalized 1:1 mix CT to the 99th percentile of the normalized 1:1 mixes of samples from healthy
volunteers.
3. Methods using CTs of the 1:1 mix, NPP, and patient’s plasma.
a. The ICA (Rosner index), where ICA ¼ [(1:1 mix CT  NPP CT)/patient CT] x 100
b. A percent correction formula, where % correction ¼ [(patient CT  1:1 mix CT)/(patient CT  NPP CT)] x 100
c. Does the 1:1 mix CT correct to more than half the difference between the patient’s neat CT and the NPP CT?

Abbreviations: CT, clotting time; ICA, index of circulating anticoagulant; NPP, normal pooled plasma.

Seminars in Thrombosis & Hemostasis Vol. 39 No. 3/2013

288 Mixing Tests in the Investigation of Prolonged PT and APTT Kershaw, Orellana

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Fig. 3 Comparison of four different formulae used in activated partial thromboplastin times (APTT) mixing test interpretation. These 1:1 mixing
tests were performed on 55 samples containing either single or multiple factor deficiencies (solid triangles) and on 44 samples comprising
inhibitors to single factors, lupus anticoagulants, therapeutic doses of unfractionated heparin, or the direct thrombin inhibitor dabigatran (open
circles). All mixing tests were performed without incubation on the STA-R analyzer (Diagnostica Stago, Asnieres, France) using TriniCLOT APTT S
(Tcoag, Bray, Ireland) as the activating reagent and a commercial lyophilized NPP (TriniCHECK Level 1 control; Tcoag) and all are immediate mixes.
Panel A shows the values obtained by subtracting the NPP CT from the 1:1 mix CT. Corrections to within 4 seconds of the NPP were consistent with
factor deficiency and more than 8 seconds difference consistent with the presence of an inhibitor. Between these values, inhibitors and factor
deficiencies could not be distinguished from one another. Data for the ratio of the 1:1 mix APTT to normal pool APTT (panel B) are remarkably
similar to panel A. Here, a ratio of below 1.1 would exclude an inhibitor and a ratio above 1.2 would exclude the presence of a factor deficiency in all
but one case of FVIII deficiency. When the mixing data were calculated for the index of circulation anticoagulant (ICA, or Rosner index) (panel C), a
level of > 11% was consistent with the presence of an inhibitor; levels below 5% were consistent with factor deficiency. The cutoff of 15% for ICA
is shown for comparison. When the percent correction formula of Chang was used (panel D), all factor-deficient samples had greater than 72%
correction, as did some inhibitor-containing samples, most noticeably the heparinized samples. A percent correction below 72% therefore
excluded factor deficiency.

the mix result is less than a chosen ratio value (e.g., 1.2); and
(iii) comparing the mix CT to the NPP CT plus 5 seconds.26
When mixing tests are performed in the context of testing
for the presence of LA testing, two choices for setting cutoff
levels have been recommended.25 The first is to determine
the 99th percentile of the distribution of healthy volunteer
plasma samples tested in 1:1 mixes with NPP. If the 1:1 mix of
the test plasma falls below the cutoff, the presence of LA is
excluded. The second method is the use of a numerical index
first proposed by Rosner et al,27 which has been known as the
Rosner Index, or the index of circulating anticoagulant (ICA).
The ICA of a test plasma is calculated from the CTs of the NPP,
the patient’s neat plasma, and the 1:1 mix of these, where
ICA ¼ [(CT 1:1 mix  CT NPP)/CT patient]  100. The origi-
nal ICA cutoff level (i.e., 15%) was taken as the upper level of
the 95% confidence interval of data from 34 plasma samples
(18 hemophiliacs and 16 inpatients defined as “normal”)
tested manually using the kaolin CT.27 Samples showing an
ICA above 15% were considered non-correcting, hence po-
tentially containing LA. However, 15% is unlikely to be the

Seminars in Thrombosis & Hemostasis Vol. 39 No. 3/2013

 Mixing Tests in the Investigation of Prolonged PT and APTT
appropriate cutoff for individual laboratories testing with
modern PT and APTT reagents by automated analysers.4 To
use the ICA most effectively, a local cutoff value based on the
specific screening test reagent/NPP/analyzer combination is
thus preferable. These cutoff levels may vary with different
test types, namely, APTT or PT. We have established a cutoff of
11% for the ICA in an APTT test system as shown in by testing
samples covering a wide range of factor deficiencies and
inhibitors that were processed under the same conditions
as the routine hemostasis work.
Another approach to interpreting mixing test results is to
calculate the “percent correction” as described by Chang
et al.28 Like the ICA, the percent correction formula incorpo-
rates the patient’s neat plasma CT, where percent correction
¼ [(patient neat CT  1:1 mix CT)/(patient neat CT  NPP
CT)]  100. With this formula, there was some overlap in the
percent correction ranges observed in factor-deficient sam-
ples and inhibitor-containing samples.28 The discriminatory
power of the formula was improved when mixes were tested
as 4-part patient plasma to 1-part NPP, with the 4:1 mix CT
replacing the 1:1 mix CT in the equation.28 By using an
adjusted cutoff of 50%, the authors observed that plasmas
with more severe factor deficiency tended to have higher
percent corrections than samples with mild factor deficiency
that had only borderline percent corrections plus some that
fell below the cutoff (false negative for factor deficiency).
We have applied four of these cutoff levels to 1:1 mixing
tests performed on 57 samples with a range of single factor
deficiencies or multiple factor deficiencies and 44 samples
with inhibitors of various types (►Fig. 3, panels A–D). All the
samples tested were surplus citrated plasma from patients
having coagulation tests performed in our laboratory. The
first observation is the overlap in 1:1 mix CTs of samples with
factor deficiency and samples containing inhibitors regard-
less of the interpretive method. This suggests no single
method of setting a cutoff can perfectly differentiate between
factor deficiencies and inhibitors in this test system. Second,
for methods that primarily use the 1:1 mix CT relative to a
fixed cutoff such as NPP þ 5 seconds or as a ratio to the NPP
Table 4 Percent of samples in the mixing test study in ►Fig. 3 showing a correct interpretation according to different methods of
evaluation and different cutoff levels
Method of evaluation Correction cutoff
(A) 1:1 mix CT minus NPP CT < 4 seconds
< 8 seconds
(B) Ratio 1:1 mix CT to NPP CT < 1.1
< 1.2
(C) ICA < 11%
< 15%
(D) Percent correction > 72%
Abbreviations: CT, clotting time; ICA, index of circulating anticoagulant; NPP, normal pooled plasma.
Notes: Factor-deficient samples (n ¼ 57) must satisfy the cutoff level to have a correct interpretation. Inhibitor-containing samples (n ¼ 44) must fall
on the opposite side of the cutoff to be called non-correcting.
Kershaw, Orellana 289
CT, the pattern of data is very similar (►Fig. 3, panels A, B).
Third, the result patterns from applying the ICA and percent
correction formula, both of which use all the available CTs,
show clear differences from each other (►Fig. 3, panels C, D).
By adjusting the cutoff levels to make all of the known factor-
deficient samples show a correction, some of the inhibitor
samples are also classified as correcting. ►Table 4 summa-
rizes the percentage of samples having correct interpreta-
tions according to different cutoffs. In the laboratory setting,
the performance of mixing tests in identifying inhibitors will
be better than that these figures suggest if additional infor-
mation is taken into account. For plasma containing heparin
or one of the direct thrombin inhibitors, the thrombin time
will be prolonged, alerting the technologists to their presence
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should this information not be otherwise available.
When evaluating the percent correction formula using
immediate 1:1 mixes in our test system, all of the 57
factor-deficient samples tested showed a greater than 72%
correction with only 2 of the 18 samples with LA correcting
more than 72% (false negative for inhibitor) (►Fig. 3, panel D).
The FVIII inhibitor samples were split evenly between correc-
tion and non-correction, as might be expected with immedi-
ate mixes as opposed to incubated mixes where the FVIII
inhibitory action is more completely expressed.
The heparinized plasmas all showed more than 72% cor-
rection (false negative for inhibitor), but in routine practice a
thrombin time test can be performed when an unexpectedly
prolonged APTT is observed to check for the possible presence
of heparin or a direct thrombin inhibitor such as dabigatran.
Occasionally, a patient has a slowly rising PT and/or APTT in
the absence of, or inconsistent with, any concomitant antico-
agulant therapy. The clinician may request a mixing test to
exclude the presence of a factor inhibitor that may have
recently arisen. In these situations the clinician may be
satisfied that a correction of the mixing test excludes an
inhibitor, without the need for further investigation. The
laboratory should be aware of the limitations of their mixing
test cutoff value in excluding inhibitors, and perhaps choose a
different cutoff for this purpose.
Correct interpretation
Factor deficient, n (%) Inhibitor, n (%)
50 (88) 44 (100)
57 (100) 34 (77)
46 (81) 44 (100)
56 (98) 36 (82)
57 (100) 33 (75)
57 (100) 27 (61)
57 (100) 26 (59)
Seminars in Thrombosis & Hemostasis Vol. 39 No. 3/2013
290 Mixing Tests in the Investigation of Prolonged PT and APTT
Conclusion
Mixing tests are an important and useful first step in the
investigation of prolonged PT and APTT screening tests. The
role of the mixing test can be simply expressed as a method to
discriminate factor deficiency from an inhibitor as the cause
of the prolonged screening test(s). The main challenge to the
laboratory is to establish reliable criteria that maximize the
chances of correct mixing test result interpretation. Several
formulae exist, from which the laboratory can choose for mix
test interpretation (►Table 3). It is a good laboratory practice
to establish local cutoff values for the analyzer, normal
plasma, and reagent types in use for whichever formula is
in use. In our own experience, no single mix test interpreta-
tion approach is 100% sensitive or specific for distinguishing
coagulation factor deficiencies or inhibitors. Our current
practice is to use the ICA for both PT and APTT immediate
1:1 mix test interpretation because it is simple to calculate
and uses all the available CTs. When the ICA is above the cutoff
value, we are confident that an inhibitor is present. If the ICA
is below the cutoff value, the most likely cause is factor(s)
deficiency, with the caveat that a weak LA or slow-acting
inhibitor is not excluded.
Thus, there is added value in the use of additional tests, such
as the thrombin time, to assist in mixing test interpretation,
particularly when the mix test result is not entirely clear.
Nevertheless, the mixing test offers substantial benefit to the
initial evaluation of abnormal CTs, as it helps to guide further
investigation toward performance of coagulation factor testing
or inhibitor testing, thereby reducing costs and time, maxi-
mizing diagnostic efficiency, and ultimately safeguarding the
quality of testing along with patient safety.29
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