Evaluation of Prolonged APTT:

The Use of Laboratory Mixing

Studies
George A. Fritsma, MS, MT(ASCP)

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.

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

The surgeon delayed the procedure until the laboratory could resolve the
prolonged PTT.

the thrombin time to well beyond

Possible Reasons for the that interval.
Prolonged PTT
Factor Deficiency or Specific
Heparin Therapy Inhibitor

When heparin is suspected, the Most congenital factor

Volume 17, Number 10 laboratory scientist may contact deficiencies cause bleeding in
the patient unit for a history. childhood. Adults rarely develop
October 2003 factor deficiencies. Acquired
However, heparin may be
Objective: The reader will be able unreported; for example, when a hemophilia is the most common of
to determine an appropriate small volume is used to flush a the acquired single factor
laboratory evaluation of an elevated line. When in doubt, the scientist deficiencies described in adults
APTT as well as differential diagno- performs the thrombin time test. that prolongs the PTT without
sis of an elevated APTT. The normal cutoff is less than 21 affecting the PT.2 It results from
seconds, and heparin prolongs the formation of autoantibodies
 CLINICAL HEMOSTASIS REVIEW specific for coagulation factor VIII. reagent plasma’s PTT, the PTT is
3176 South Peoria Court Anti-VIII autoantibodies may arise said to be “corrected.” In this
Aurora, CO 80014 in pregnancy, autoimmune instance, the patient may have a
520.722.0797
disorders, or in the elderly, causing congenital factor deficiency. This is
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severe, often life-threatening confirmed by performing a series of
CLINICAL ADVISORS hemorrhage.3 factor assays, first VIII, then IX,
Dorothy M. Adcock, MD and then XI, the three most likely
Alexander Duncan, MD, ChB deficiencies. Most acute care
H. James Day, MD, FACP Lupus Anticoagulant hemostasis laboratories are
Don W. Hill, MD, FACP equipped to measure these factors.
Lupus anticoagulants (LACs)
Alternatively, the patient may have
CONTRIBUTOR are autoantibodies that react with
Lynne Stevens, MT(ASCP)
a temperature- and time-dependent
phospholipid-bound proteins. LACs
anti-VIII inhibitor. The inhibitor may
are present in 1-2% of unselected
then be a warm reacting IgG
individuals.4 LACs cause
antibody that requires one or two
prolongation of phospholipid-
hour’s incubation at 37°C to be
dependent laboratory assays,
detected. If the incubated mixture
particularly the PTT, and may also
does not correct, meaning if the
be detected in immunoassay
PTT is at least 15% longer than the
systems such as the anti-B-2
normal reagent plasma’s incubated
glycoprotein I, anti-prothrombin, or
PTT, anti-VIII is suspected. A low
anti-cardiolipin antibody assay.
factor VIII activity level further
Most LACs appear in response to
suggests this possibility. The
inflammation, have no clinical
Clinical Hemostasis Review is published presence of a factor VIII inhibitor is
by Esoterix Coagulation, Inc. and is circu- effects, and disappear in 6-8
confirmed by a Bethesda titer,
lated to selected physicians and labora- weeks. Chronic LACs, however, are
usually performed by a specialty or
torians. Copyright 2003. Esoterix Coagu- associated with a risk of venous
lation is part of the ESOTERIX, Inc. family of reference hemostasis laboratory.
laboratories providing exoteric testing in
thrombosis, strokes, myocardial
numerous disease corridors. The opinions infarctions, peripheral artery
expressed in the articles are those of the occlusion, and recurring No Correction Upon
author(s) and do not necessarily reflect the spontaneous abortions. LACs Immediate Testing
opinions or recommendations of the adver-
tisers, editors, or publisher. The publisher rarely cause bleeding. If the PTT result fails to
reserves copyright and renewal on all pub- normalize or correct upon
lished material and such material may not be immediate testing, an inhibitor is
reproduced in whole or in part without writ-
ten permission from the publisher. Consult How to Decide: Mixing present. The most likely possibility
the full prescribing information on any drugs is LAC. Anti-VIII is still in the
or devices discussed. Studies differential, as some VIII inhibitors
All correspondence should be directed
Acute care laboratories must be can react immediately, however,
to the attention of the Editor, Clinical Hemo-
stasis Review, 3176 South Peoria Court, equipped to perform PTT mixing anti-VIII is unlikely in the absence
Aurora, CO 80014. studies. Mixing studies distinguish of bleeding. When chronic, LAC is
Subscription Rate: $65.00/year, 14 is- associated with thrombosis.5
sues, prepaid. Outside the USA additional
between factor deficiencies and the
postage is required: Canada $20.00/year, presence of inhibitors; also
all other destinations $50.00/year. Single between specific inhibitors and Lupus Anticoagulant
copies $7.00. Subscriptions not accompa- LACs. The laboratory scientist
nied by payment will be assessed a billing
prepares a 1:1 mixture of patient’s
Testing
charge.
To assure prompt delivery of your is- platelet-poor plasma and normal Many acute care facilities prefer
sues, please notify us of any address correc- reagent platelet-poor plasma, and to order LAC testing from specialty
tions. Six weeks are required to effect a performs a PTT on the mixture. The laboratories, as the diagnosis
change. Missing copies not received by mail
will be provided free of charge if we are normal reagent platelet-poor requires a complex series of
notified no later than two months after the plasma may be purchased from a reflexive assays and a
issue date. After this deadline, we will commercial distributor or prepared pathologist’s interpretation.
charge subscribers $5.00 per issue. locally from a normal donor. Two test systems are necessary,
ISSN 0894-1025
owing to the heterogeneity of
Correction upon LACs.6 The first uses a specially
Immediate Testing prepared PTT reagent that is
sensitive to LAC. The second
If the mixture yields a PTT employs the dilute Russell’s viper
result within 10% of the normal venom time assay, also sensitive
to LAC, even in instances where

2 CLINICAL HEMOSTASIS REVIEW / OCTOBER 2003

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

4 CLINICAL HEMOSTASIS REVIEW / OCTOBER 2003

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

OCTOBER 2003 / CLINICAL HEMOSTASIS REVIEW 5

• Mixing studies may be 4. Bevers EM, et al. Lupus
anticoagulant IgG’s are not
employed any time the initial
directed to phospholipids only, but
PTT result exceeds the upper
to a complex of lipid-bound human
limit of the reference interval. A
prothrombin. Thromb Haemostas
mix is said to correct when the
1991; 66: 629-632.
result of the mixture exceeds
the result of the normal plasma
5. Goldsmith JC. Diagnosis of
by no more than 10%. A mix is
factor VIII versus nonspecific
uncorrected when the result
inhibitors. Semin Hematol 1993; 30
exceeds the normal plasma
(Suppl 1): 3-6.
result by more than 15%.
Results in the 10-15% range
6. Brandt JT, et al. Criteria for the
are equivocal and require
diagnosis of lupus anticoagulants:
repeating. If a laboratory is
an update. Thromb Haemostas
getting a large number of
1995; 74: 1185-1190.
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.

6 CLINICAL HEMOSTASIS REVIEW / OCTOBER 2003