1) A 63-year-old man had a prolonged PTT prior to hip surgery. Mixing studies showed no correction, indicating an inhibitor was present. Further testing confirmed the presence of a lupus anticoagulant. 2) A 31-year-old woman experienced bleeding after delivery. Her PTT was prolonged while her PT and platelet count were normal, ruling out DIC. Mixing studies showed no correction upon incubation, indicating an acquired factor VIII inhibitor, which was confirmed. 3) The document discusses evaluating and differentially diagnosing an elevated PTT through mixing studies and additional targeted testing to distinguish between factor deficiencies, inhibitors, and lupus anticoagulants.

1. Objective: The reader will be able
to determine an appropriate
laboratory evaluation of an elevated
APTT as well as differential diagno-
sis of an elevatedAPTT.
Evaluation of Prolonged APTT:
The Use of Laboratory Mixing
Studies
George A. Fritsma, MS, MT(ASCP)
Volume 17, Number 10
October 2003
A Case of Pre-Operative PTT Prolongation
A 63-year-old man was scheduled for hip replacement surgery. He
had no history of bleeding or thrombosis, was taking no anticoagulants,
and was in general good health. The surgeon ordered a prothrombin time
(PT) and partial thromboplastin time (PTT) as pre-operative screens.1
The results are given in table 1.
The surgeon delayed the procedure until the laboratory could resolve the
prolonged PTT.
Possible Reasons for the
Prolonged PTT
Heparin Therapy
When heparin is suspected, the
laboratory scientist may contact
the patient unit for a history.
However, heparin may be
unreported; for example, when a
small volume is used to flush a
line. When in doubt, the scientist
performs the thrombin time test.
Factor Deficiency or Specific
Inhibitor
Most congenital factor
deficiencies cause bleeding in
childhood.Adults rarely develop
factor deficiencies.Acquired
hemophilia is the most common of
the acquired single factor
deficiencies described in adults
that prolongs the PTT without
affecting the PT.2
It results from
the formation of autoantibodies
The normal cutoff is less than 21
seconds, and heparin prolongs
the thrombin time to well beyond
that interval.
Table #1: Pre-operative PT and PTT Results
Test Patient Result Reference Interval
PT 14.1 seconds (INR 1.1) 12.4-14.4 seconds
PTT 68 seconds 25-34 seconds

2. 2 CLINICAL HEMOSTASIS REVIEW / OCTOBER 2003
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H. James Day, MD, FACP
Don W. Hill, MD, FACP
CONTRIBUTOR
Lynne Stevens, MT(ASCP)
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specific for coagulation factor VIII.
Anti-VIII autoantibodies may arise
in pregnancy, autoimmune
disorders, or in the elderly, causing
severe, often life-threatening
hemorrhage.3
Lupus Anticoagulant
Lupus anticoagulants (LACs)
are autoantibodies that react with
phospholipid-bound proteins. LACs
are present in 1-2% of unselected
individuals.4
LACs cause
prolongation of phospholipid-
dependent laboratory assays,
particularly the PTT, and may also
be detected in immunoassay
systems such as the anti-B-2
glycoprotein I, anti-prothrombin, or
anti-cardiolipin antibody assay.
Most LACs appear in response to
inflammation, have no clinical
effects, and disappear in 6-8
weeks. Chronic LACs, however, are
associated with a risk of venous
thrombosis, strokes, myocardial
infarctions, peripheral artery
occlusion, and recurring
spontaneous abortions. LACs
rarely cause bleeding.
How to Decide: Mixing
Studies
Acute care laboratories must be
equipped to perform PTT mixing
studies. Mixing studies distinguish
between factor deficiencies and the
presence of inhibitors; also
between specific inhibitors and
LACs. The laboratory scientist
prepares a 1:1 mixture of patient’s
platelet-poor plasma and normal
reagent platelet-poor plasma, and
performs a PTT on the mixture. The
normal reagent platelet-poor
plasma may be purchased from a
commercial distributor or prepared
locally from a normal donor.
Correction upon
Immediate Testing
If the mixture yields a PTT
result within 10% of the normal
reagent plasma’s PTT, the PTT is
said to be “corrected.” In this
instance, the patient may have a
congenital factor deficiency. This is
confirmed by performing a series of
factor assays, first VIII, then IX,
and then XI, the three most likely
deficiencies. Most acute care
hemostasis laboratories are
equipped to measure these factors.
Alternatively, the patient may have
a temperature- and time-dependent
anti-VIII inhibitor. The inhibitor may
then be a warm reacting IgG
antibody that requires one or two
hour’s incubation at 37°C to be
detected. If the incubated mixture
does not correct, meaning if the
PTT is at least 15% longer than the
normal reagent plasma’s incubated
PTT, anti-VIII is suspected. A low
factor VIII activity level further
suggests this possibility. The
presence of a factor VIII inhibitor is
confirmed by a Bethesda titer,
usually performed by a specialty or
reference hemostasis laboratory.
No Correction Upon
Immediate Testing
If the PTT result fails to
normalize or correct upon
immediate testing, an inhibitor is
present. The most likely possibility
is LAC. Anti-VIII is still in the
differential, as some VIII inhibitors
can react immediately, however,
anti-VIII is unlikely in the absence
of bleeding. When chronic, LAC is
associated with thrombosis.5
Lupus Anticoagulant
Testing
Many acute care facilities prefer
to order LAC testing from specialty
laboratories, as the diagnosis
requires a complex series of
reflexive assays and a
pathologist’s interpretation.
Two test systems are necessary,
owing to the heterogeneity of
LACs.6
The first uses a specially
prepared PTT reagent that is
sensitive to LAC. The second
employs the dilute Russell’s viper
venom time assay, also sensitive
to LAC, even in instances where

3. 4 CLINICAL HEMOSTASIS REVIEW / OCTOBER 2003
the PTT system fails. Lack of
correction in mixing studies of
either abnormal test is presumptive
evidence for LAC. These results
are followed with neutralization
studies using high phospholipid
reagents. Correction by the
neutralizing reagents confirms the
presence of LAC.
Lupus anticoagulants are part
of a family of antibodies called anti-
phospholipid antibodies. These
may also be detected using a
series of immunoassays:
· Anticardiolipin IgG, IgM, or
IgA antibody
· Antiphosphatidyl serine
IgG or IgM antibody
· Anti-b2 glycoprotein I IgG
or IgM antibody
· Anti-prothrombin IgG or
IgM antibody
Like the selection of lupus
anticoagulant assays, the
selection of an anti-phospholipid
antibody immunoassay is made in
consultation with a specialty
laboratory pathologist. Profiles
consisting of several of these
immunoassays are recommended.
A Case of Pre-Operative
PTT Prolongation:
Conclusion
Mixing studies were performed
in follow-up to the prolonged PTT
result as shown in Table 2.
The thrombin time confirmed
that the patient had received no
heparin. The mixing study results
indicate an inhibitor. LAC was first
in the differential because the
patient was experiencing no
bleeding. The results from the
reference laboratory confirmed the
presence of LAC both in the PTT-
and dRVVT-based assays.
Surgery was successful and the
patient was prescribed Coumadin®
post-surgically to reduce the risk of
thromboembolic disease.A PT and
PTT performed after Coumadin®
treatment was completed were
normal, indicating that the LAC
was transient.
A Case of Post-Partum
Hemorrhage
A 31-year-old woman
experienced vaginal bleeding,
nosebleeds, and bruising one week
after a normal delivery. She had no
previous history of bleeding, no
bleeding in her kindred, and was
taking no anticoagulant drugs. Her
gynecologist ordered a PT, PTT,
and complete blood count. The
results for the PT, PTT, and platelet
count are shown in Table #3.
As in the first case, the differential
for this patient includes therapeutic
heparin, factor deficiency, acquired
factor inhibitor, or lupus
anticoagulant. Disseminated
intravascular coagulation and von
Willebrand disease are further
possibilities.
Table #4: Expected results in DIC
Table #3: PT, PTT, and platelet count results in a woman one week
after delivery
Table #2: Results of PTT Mixing
Studies
Disseminated
intravascular coagulation
Disseminated intravascular
coagulation (DIC) is uncontrolled
activation of the coagulation
mechanism. DIC is the immediate
cause of death in many cases of
inflammation and infection, and the
core hemostasis laboratory must
be equipped to diagnose it and
monitor its treatment.In DIC,
platelets and coagulation factors
are consumed, causing
thrombocytopenia, prolonged PT
and PTT, and reduced fibrinogen
levels. Further, fibrinolysis
becomes activated, producing a
variety of peptides called fibrin
degradation products. One such
product is D-dimer. The laboratory
must be equipped to measure D-
dimer levels, either through the
time-honored latex agglutination
assay or the more recently
available quantitative D-dimer
methods. The recommended tests
for DIC and their expected results
are given in Table 4.
Test Result Reference Interval
Thrombin time 17.5 seconds < 21 seconds
Patient PTT 68 seconds 25-34 seconds
PTT reagent normal plasma 29 seconds Commercial platelet poor
plasma
1:1 mixture 56 seconds No correction (> 15% above
PTT reagent normal plasma,
or > 33.4 seconds)
Test Result Reference Interval
PT 14.1 seconds (INR 1.1) 12.4-14.4 seconds
PTT 82 seconds 25-34 seconds
Platelet count 279 x 109
/L 140-450 x 109
/L
Test Anticipated Value in DIC
Platelet count < 150 x 109
/L
D-dimer qualitative >1:2
D-dimer quantitative > 500 ng/mL
Fibrinogen < 200 mg/dL
PTT Prolonged

4. OCTOBER 2003 / CLINICAL HEMOSTASIS REVIEW 5
DIC treatment is monitored by
serial platelet counts, D-dimer, and
the fibrinogen assay. In the case of
the 31 YO woman, DIC is unlikely
as the PT and platelet count are
normal. Nevertheless, because of
the profound implications of DIC, a
D-dimer and fibrinogen were
performed as part of the follow-up
laboratory assay. See table #5.
Von Willebrand Disease
Sometimes bleeding occurs
when the PT and PTT are normal
and the platelet count exceeds
50x109
/L. Von Willebrand disease
(vWD), with a prevalence
exceeding 0.1% worldwide, is the
most likely explanation.
Since von Willebrand factor is
the carrier protein for factor VIII,
low factor VIII activity,
accompanied by a slightly
prolonged PTT, may also point to
vWD. When von Willebrand
disease is suspected, the core
laboratory should request a von
Willebrand profile from a reference
or specialty laboratory.
In this case, the von Willebrand
diagnosis is secondary, as the
elevated estrogens in pregnancy
raise the level of von Willebrand
factor and reduce the bleeding
symptoms. Further, because von
Willebrand disease is inherited it is
likely to appear with symptoms in
childhood, and may be found in
other members of the family.
Nevertheless, a von Willebrand
disease profile was ordered from a
reference laboratory. The results
demonstrated that the diagnosis
was unlikely.
A Case of Post-partum
Hemorrhage: Conclusion
Mixing studies were performed
in follow-up to the prolonged PTT
result as shown in Table # 5.
The thrombin time confirmed
that the patient had received no
heparin. The normal
fibrinogen and D-dimer levels rule
out DIC. The mixing study results
indicate an inhibitor. Factor VIII
inhibitor was first in the differential
because the patient was
experiencing bleeding. The results
of the incubated mixing study
implied the presence of a warm-
reacting antibody which was
confirmed by the Bethesda titer.
Acquired hemophilia is transient
but often life-threatening. The
patient was treated with an
activated prothrombin complex
concentrate, factor eight inhibitor
bypassing activity (FEIBA®) and
monitored with repeat factor VIII
and Bethesda titers until the
inhibitor could no longer be
demonstrated.
Conclusion and Further
Recommendations
• Mixing studies may be
necessary any time there is a
prolonged PTT in the absence
of reported heparin therapy.
• Mixing studies that include
incubation are used to
distinguish among single and
multiple factor deficiencies,
coagulation factor inhibitors,
and lupus anticoagulant.
• Mixing studies are effective
when the patient plasma tested
is platelet-poor plasma with a
platelet count of less than 10 x
109
platelets per mL. To make
platelet-poor plasma, centrifuge
the specimen at 2500 x g for
10 minutes. Transfer the
plasma with a plastic pipette
into a plastic centrifuge tube,
cap and centrifuge an
additional 10 minutes at 2500 x
g. Excessive numbers of
plasma platelets will partially
neutralize lupus anticoagulant
in vitro, secrete excessive
levels of stored coagulation
factors, and partially neutralize
heparin in vitro. The platelet
poor plasma centrifugation
method requires routine
monitoring.
• Mixing studies usually use
equal portions of patient and
normal platelet poor plasma.
For increased sensitivity to
weak inhibitors, a 4:1 ratio of
patient to normal plasma may
be used.
Table #5: Final Laboratory workup of post-partum
hemorrhage
Test Result Reference Interval
Thrombin time 18 seconds < 21 seconds
Fibrinogen 324 mg/dL 226-467 mg/dL
D-dimer quantitative 146 ng/mL < 240 ng/mL
Patient PTT 82 seconds 25-34 seconds
PTT reagent normal plasma 29 seconds Commercial platelet
poor plasma
1:1 mixture 33 seconds Correction
PTT reagent normal plasma 41 seconds Commercial platelet
incubated 2 hours at 37ºC poor plasma
1:1 mixture incubated 67 seconds No correction
2 hours at 37ºC (> 47 seconds)
Factor VIII < 1% Implies acquired
hemophilia
Bethesda titer 38 Bethesda units Autoimmune inhibitor
to factor VIII

5. 6 CLINICAL HEMOSTASIS REVIEW / OCTOBER 2003
• Mixing studies may be
employed any time the initial
PTT result exceeds the upper
limit of the reference interval.A
mix is said to correct when the
result of the mixture exceeds
the result of the normal plasma
by no more than 10%. A mix is
uncorrected when the result
exceeds the normal plasma
result by more than 15%.
Results in the 10-15% range
are equivocal and require
repeating. If a laboratory is
getting a large number of
equivocal results, it may be
that the specimens are not
sufficiently platelet poor.
Separate cutoff limits are
determined through empirical
observation when a 4:1 or other
mix is used.
• Though less common, mixing
studies may also be employed
in the PT or thrombin time test
system when appropriate.
• All coagulation laboratories,
large and small, should be
equipped to perform mixing
studies.
References
1. Eisenberg J, Clarke JR,
Sussman SA. Prothrombin and
partial thromboplastin times as
preoperative screening tests.Arch
Surg 1982; 117:48.
2. Boggio LN, Green D.Acquired
hemophilia. Rev Clin Exp Hematol
2001; 5: 889-404.
1185-1190.
3. Ludlam DA, Morrison AE,
Kessler C. Treatment of acquired
hemophilia. Semin Hematol 1994;
31 (Suppl 4) 16-19.
4. Bevers EM, et al. Lupus
anticoagulant IgG’s are not
directed to phospholipids only, but
to a complex of lipid-bound human
prothrombin. Thromb Haemostas
1991; 66: 629-632.
5. Goldsmith JC. Diagnosis of
factor VIII versus nonspecific
inhibitors. Semin Hematol 1993; 30
(Suppl 1): 3-6.
6. Brandt JT, et al. Criteria for the
diagnosis of lupus anticoagulants:
an update. Thromb Haemostas
1995; 74: 1185-1190.