SOLUTIONS ANTIPHOSPHOLIPID N 3 / 2012

No 3 / 2012
SOLUTIONS
THE BULLETIN OF GAMMA-DYNACARE LABORATORIES
ANTIPHOSPHOLIPID
SYNDROME:
DIAGNOSING A
COMPLEX CONDITION
Antiphospholipid syndrome (APS),
sometimes known as Hughes Syndrome,
is an autoimmune disease with serious
ramifications including venous/arterial
thrombosis, and pregnancy complications
such as recurrent fetal loss and severe preeclampsia. Sera taken from patients with
APS often contain antibodies to cardiolipin,
an acidic phospholipid - hence its original
name “antiphospholipid syndrome”. To this
day, APS remains a complex illness that is
inherently difficult to diagnose.1,2
It is believed that APS affects between
1–5% of the population. APS is a major
women’s health issue, as 75–90%
affected are women. It is estimated APS
is the cause of 15–20% of all thrombotic
events, 10–25% of recurrent miscarriages,
and one-third of the strokes in patients
under the age of 50. The syndrome is
also thought to be present in 40–50% of
patients with systemic lupus erythematosus
(SLE).3,4
Pathogenesis of APS
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Thrombosis has a key role in the
clinical manifestations of APS. Several
mechanisms have been proposed for
APS-related thrombosis; however, it is
most probably multifactorial in etiology.
The thrombotic tendency may be caused
by antiphospholid antibodies through the
following mechanisms:2
pInhibition of the factors of the
anticoagulant system, affecting
thrombin formation and antithrombin
activity.
pImpairment of fibrinolytic activity.
pInterference with coagulation factors
and complement, particularly the
intrinsic and protein C pathway.
pDirect effect of antiphospholipid
antibodies on cell function, such as
platelets, endothelial cells and
vascular cells.
There is growing evidence that
phospholipid antibodies are present in
patients months to years before the onset
of any clinical symptoms of APS, possibly
induced by exogenous stimulus or due to
antigen spreading.3,5 Many individuals with
APS may never experience a thrombotic
event or pregnancy loss.5 Secondary risk
factors, co-morbidities, and other factors
influence whether a patient actually
experiences the clinical manifestations
of APS. Thrombosis may be initiated by
trigger events, such as surgery, pregnancy,
or the use of oral contraceptives.3
Thrombotic events can affect arteries
and veins of all sizes, including the
microvascular system. The thrombosis
can also be recurrent.3 The most
frequent thrombotic events are deep-vein
thrombosis (DVT) and ischemic stroke
(IS). Catastrophic APS is a very rare
variant occurring in less than 1% of all
APS patients. This is an accelerated form
of this syndrome that results in multiple
organ failure.2, 3
SOLUTIONS
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Diagnosis
The International Consensus Statement
for the Classification Criteria for Definite
Antiphospholipid Syndrome was
updated in 2006 to aid physicians in
the diagnosis.2,6 Unfortunately, despite
these improved guidelines, diagnosing
APS in the clinical setting still remains a
challenge.
The Sapporo classification criteria
were first formulated and published in
1999.2,6 The revised criteria, now known
as the Sydney criteria, were proposed
at a workshop in Sydney, Australia at
the Eleventh International Congress
on antiphospholipid antibodies. It was
proposed that a patient must have at
least one clinical criterion and one
positive laboratory criterion to be
diagnosed with APS.6
pThe elapsed time of 12 weeks between
the initial and confirmatory test is to
increase the probability of excluding
temporary infection-associated
antibodies.2
pIf there are fewer than 12 weeks, or more
than 5 years between a positive APS
test and the clinical manifestations, the
classification criteria should not be used.6
The panel discussed clinical and
laboratory features associated with APS
that are not included in the revised
criteria.3,6 The committee decided that if
included, it may decrease diagnostic
specificity. These include:
pHeart valve abnormalities
pLivedo reticularis
pThrombocytopenia
pNephropathy
Clinical criteria6
pVascular thrombosis:
PAt least one confirmed clinical episode
in an arterial, venous, or small vessel
in any tissue or organ that has been
confirmed.
pPregnancy morbidity:
POne or more unexplained deaths of a
normal fetus at or beyond the 10th week
of gestation.
PThree or more unexplained consecutive
spontaneous abortions before the 10th
week of gestation.
POne or more premature births of a
normal neonate delivered prematurely
before the 34th week of gestation due
to severe pre-eclampsia, eclampsia, or
severe placental insufficiency (where
other causes are excluded).
Laboratory criteria6
pPositive results on two or more
occasions at least 12 weeks apart for at
least one of the following tests:
PLupus anticoagulant (LA);
PCardiolipin antibodies (aCL) of IgG and/
or IgM isotype in medium or high titre,
> 40 units;
Pß2 -glycoprotein I (ß2GPI) antibodies of
IgG and/or IgM isotype.
pNeurological manifestations
pIgA Cardiolipin antibodies
pIgA ß2 -glycoprotein I antibodies
pPhosphatidylserine antibodies
pPhosphatidylethanolamine antibodies
pProthrombin antibodies
pPhosphatidylserine-prothrombin
complex antibodies
Although the pathophysiology of APS is
now relatively well understood, difficulties
still persist with making a definitive
diagnosis based solely on clinical criteria.
The prevalence of the clinical symptoms
is high and the differentials for vascular
thrombosis and pregnancy morbidity are
relatively broad. Laboratory testing can
therefore be instrumental in providing
definitive diagnosis.
APS laboratory testing
Laboratory testing for APS includes
both functional coagulation assays
and immunology testing.2 Lupus anticoagulant (LAC) testing is an in vitro
functional assay detecting the effect
phospholipid antibodies have on the
coagulation cascade. Phospholipid
antibody tests are a direct measure of the
presence or absence of the antibodies
themselves. There are two main classes
of antiphospholipid antibodies assays
available. Cardiolipin antibodies detect
antibodies binding to ß2 -glycoprotein
within context of a protein/phospholipid
complex whereas ß2 -glycoprotein I
antibodies detect antibodies binding to
ß2-glycoprotein in absence of phospholipids.2
Predicting future
APS-associated
events
Several studies have
been done on the use
of these laboratory tests
in identifying patients at
risk for thrombotic events
or severe pregnancy
complications:
pPositive antiphospholipid
lab tests, in the absence
of clinical criteria, should
only be considered to be
at risk factors rather than
diagnostic criteria for
APS. Patients should be
assessed for additional
thromboembolic risk
factors (e.g., smoking,
obesity, hypertension) for
risk reduction.5
pClinical studies have
shown that testing for
the antibody profile (see
Sidebar 2) is more useful
in identifying thrombotic
risk than the result of any
individual test.2
pThe presence of both
aCL–IgG antibodies
and ß2GPI antibodies
is believed to identify
patients at higher risk for
APS.2
pThe presence of LAC
has been observed more
frequently in patients
without clinical events,
and may be false positive
in the elderly.2
pß2GPI antibodies
have been correlated
with thrombosis,
pre-eclampsia, and
eclampsia as well as
having a role in young
women with ischemic
stroke.2
SOLUTIONS
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Lupus anticoagulants (LAC)
Cardiolipin (aCL) antibodies
The term “lupus anticoagulant” was first
coined in 1972 to describe an inhibitor
directed against the coagulation cascade
phospholipids.2 The name is a misnomer
as most people testing positive for “lupus
anticoagulant” (LAC) do not have SLE
and it has a procoagulant effect in
vivo, however the name persists despite
attempts to modify.
The real limitation of the aCL assay is
inter-assay variability. The results for
different assays on the market particularly
differ in the lower measuring range for the
test.1,6,7
The LAC test detects various APA’s on
the basis of their interference with the
phospholipid dependent steps in the
coagulation cascade. Testing for the
presence of LAC is performed following
strict guidelines set up by International
Society of Thrombosis and Haemostasis
(ISTH) which include the preparation
of platelet-poor plasma, and utilizing
screening, mixing and confirmation
tests.2,7
The ISTH guidelines state that
laboratories are to perform two different
screening tests.7 They recommend
to perform (a) the dilute Russell’s
Viper Venom Time (dRVVT) and (b)
the activated partial thromboplastin
time (aPTT) using a reagent with low
phospholipid content.2 If either test is
positive, the results are to be confirmed
using a bilayer or hexagonal-phase
phospholipid based reagent.
Interpreting aCL antibodies test results
pOnly medium and high levels, > 40
units of aCL antibodies (IgG or IgM) are
included in the diagnostic criteria which
improve the specificity.2,6
pLupus anticoagulant
paCL IgA is of little diagnostic value; it
has more use in classifying patients
into diagnostic subgroups for risk since
IgA aCL may also be associated with
thrombocytopenia, skin ulcers and
vasculitis.1,6
pß2GPI glycoprotein I
antibodies, IgG and/or
IgM
ß2 -glycoprotein I (ß2GPI) antibodies
ß2GPI antibodies have been incorporated
into the 2006 updated criteria and are
considered to be the most clinically
significant antibodies. The assay shows
higher specificity than the aCL assay and
can be the only positive test in 3–10% of
the APS patients.3,6
Interpreting ß2GPI antibodies test results
pIt has been shown that LAC is
more consistent with the clinical
manifestations of APS than
phospholipid antibodies.1,5
pHigh titres of ß2GPI antibodies
are associated with a high risk of
thrombosis.6
Phospholipid antibodies
The challenge with the laboratory tests for
phospholipid antibodies (APA) is that there
is no diagnostic “gold standard”.2,5 The
presence of APA is a necessary inclusion
criterion to make the diagnosis, but is
not diagnostic of APS. The antibodies
may also be found in children with viral
infection, other autoimmune disorders,
patients with infections, malignancy and
even in healthy individuals.2
Patients presenting
with clinical symptoms
suggestive of APS should
have the following tests
ordered, particularly
those without other
common risk factors:
pAt lower concentrations, aCL-IgM
tends to give false-positive results for
APS, particularly in the presence of
rheumatoid factor or cryoglobulins.6
Interpreting LAC test results
pTesting should not be performed while
patients are on anticoagulant therapy.
Heparin and vitamin K antagonist
treatments may impair the detection of
the lupus anticoagulant.7
Tests to Order
in Cases of
Suspected APS
pInterferences by cryoglobulins or
rheumatoid factor may cause a false
positive in interpretation of IgM ß2GPI
antibodies.6
pCardiolipin antibodies,
IgG and/or IgM
Results of these tests
will be adjunctive to
the clinical findings
and should be not be
considered diagnostic
of, but rather as risk
factors for thrombosis,
pregnancy loss and
clinical manifestations
of APS.2,5 If positive,
confirmatory tests should
be repeated within 6 to
12 weeks.2,6
SOLUTIONS
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References
1.Tincani A, Andreoli L, Casu C, et al.
Antiphospholipid antibody profile:
implications for the evaluation and
management of patients. Lupus.
2010;19(4):432–435.
2.Devreese K, Hoylaerts M. Challenges
in the diagnosis of antiphospholipid
syndrome. Clin Chem. 2010:56(6):930–
940.
3.Lockshin MD, Derksen RHWM. New
developments in lupus-associated
antiphospholipid syndrome. Lupus.
2008;17(5):443–446.
4.APS Foundation of America, Inc.
Antiphospholipid antibody syndrome.
Available at: http://www.apsfa.org/aps.
htm. Accessed June 21, 2010.
5.Roubey RAS. Risky business: the
interpretation, use, and abuse of
antiphospholipid antibody tests in clinical
practice. Lupus. 2010;19(4):440–445.
6.Miyakis S, Lockshin MD, Atsumi T, et al.
International consensus statement on an
update of the classification criteria for
definite antiphospholipid syndrome (APS).
J Thromb Haemost. 2006;4(2):295–306.
7.Pengo V, Tripodi A, Reber G, et al. Update
for the guidelines for lupus anticoagulant
detection. J Thromb Haemost.
2009;7(10):1737–1740.
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