Antiphospholipid
Syndrome
Tools to Aid in the Accurate Diagnosis of
Antiphospholipid Syndrome

Redefining Autoimmunity

Antiphospholipid Syndrome
Experts in autoimmunity
Inova Diagnostics provides a comprehensive menu of products to aid in the diagnosis of Antiphospholipid Syndrome.
QUANTA Lite® ELISA tests are high quality reagents which are compatible with a variety of automation solutions offeredthrough Inova Diagnostics. QUANTA Flash® tests are chemiluminescent based reagents utilized on the BIO-FLASH®
system. QUANTA Flash reagents offer a number of advantages including broader analytical measurement range, higher
sensitivity and precise quantitation, giving clinicians added clarity in the diagnosis of APS.
Inova Diagnostics offers the most advanced APS tests including QUANTA Lite PS/PT and QUANTA Flash β2GP1 Domain 1.
In cases where LAC (lupus anticoagulant) testing results are ambiguous but the suspicion of APS is high, the presenceof anti-PS/PT antibodies may greatly aid in clarifying the diagnosis due to high correlation of these antibodies to LAC.
When PT (prothrombin) is bound to PS (phosphatidylserine), it's orientation changes and reveals clinically relevant
epitopes distinct from assays that test for PS or PT in isolation. Antibodies to PS and PT lack specificity for APS disease
manifestations while antibodies to the PS/PT complex correlate very well with both thrombotic and pregnancy
complications in APS.1-6
The QUANTA Flash β2GP1 Domain 1 assay, unique to Inova Diagnostics, identifies antibodies to domain 1 of β2GP1, theimmunodominant epitope in APS. The presence of anti-β2GP1 domain 1 antibodies is associated with an increased risk
for adverse events in APS including thrombosis.7-8

The domain 1 region of β2 glycoprotein1 (ß2GP1)
Circular form withdomain 1 hidden"J" form withdomain 1 exposed

domain 1 region

negatively charged
phospholipids

Potential Testing Algorithm for APS
Suspicion of APS

High Risk
High risk for recurring thrombosis
Moderate to High RiskaCL (IgG/IgM)
β2GPI (IgM/IgG)
LAC

Moderate Risk
Possible Risk
Moderate risk for recurring thrombosis
Unlikely

Triple positive

One or two positive

Negative

Likelihood of APS
is high

Likelihood of APS ismoderate

APS unlikely

PS/PT (IgG/IgM) and β2GP1 Domain 1

PS/PT (IgG/IgM)

β2GPI Domain 1

Any Positive
Likelihood of APS ismoderate to highPositive

Negative

Likelihood of APS ismoderateaCL IgA, β2GP1 IgANegative
Likelihood of APS ismoderate

Additional testingin cases of highclinical suspicion

Positive

Negative

Positive

Negative

High risk forrecurring thrombosis

Moderaterisk for recurring
thrombosis

APS is possible

APS unlikely

Likelihood of APS
based on testing outcome
High
Moderate to highRisk outcome• Triple positivity is the strongest risk factor for clinical manifestations2-6, 9• Titers and isotype need to be considered4-6• Inclusion of PS/PT results in excellent diagnostic accuracy and a predictor of adverse events2-6, 9Moderate• PS/PT may identify patients with APS who are negative for serological criteria markers10• Positivity for at least one of the criteria markers increases likelihood for APS• Titers and isotype need to be considered (Antibodies of IgG isotype represent higher risk than that of IgM, and higher antibody level implies higher risk)4-6

Possible• All criteria markers negative but one or more esoteric marker positive; APS possible, but likelihood only marginally increased11-12• Isolated IgA antibody positivity has been described in association with APS9, 13-14

Unlikely• All markers are negative which makes APS unlikely, but can not be excluded11-12

Important assays you should know about
Presence of anti-PS/PT IgG or IgM antibodies
indicates a higher risk category of patients

Incrementaldetection
detection rates
Incremental
ratesforfor
aPL
serologicalmarkers
inprimaryaPL serological markers in primaryAPS patientspatients (N=107)1APS(N=107)1

Antiphospholipid antibodies are not directed against
phospholipids, but rather to phospholipid-protein
complexes.
Antibodies to PS/PT correlate highly to LAC. In cases where
LAC testing results are ambiguous but the suspicion of APS
is high, the presence of these antibodies may greatly aid
in clarifying the diagnosis. Recent evidence suggests that
antibodies to the complex of PS and PT (PS/PT) identifies
a distinct subset of patients with very high likelihood ofadverse events.2 Antibodies targeted to either PS or PT
yield less clinically relevant information as antibodies
towards the PS/PT complex.2
Recent studies have shown that the risk of thrombotic
events increases with the number of positive test resultsin APS patients and aPL carriers.2 Triple positivity for
LAC, anti-β2GP1, and PS/PT demonstrated the highest
diagnostic accuracy out of 23 possible combinations ofaPL tests.2

80%
70%

20.5%

60%
50%
40%
30%

11.2%
42.1%

20%
10%
0%

ACA IgG/M/A

The domain 1 region of β2GP1 does a better job
differentiating APS from other diseases comparedto the whole molecule or domain 4/5.
3 500

Anti-β2GP1 antibodies target multiple epitopes in thesame molecule. A growing body of evidence indicatesthat domain 1 is the most relevant epitope targetedby anti-β2GP1 antibodies in patients with APS. Many
studies have shown that anti-domain 1 antibodies areassociated with aPL related events, both vascular andobstetric in nature.7

PS/PT IgG/M

Sensitivity increase as assays are added

4 000

Anti-β2GP1 domain 1 antibodies are highly specificfor the diagnosis of APS and may help support
therapeutic decision making

β2GP1 IgG/M/A

3 000
2 500

^

*
β2 GP1#β2 GP1-D1(whole molecule)

^

#

AD
children
n=33

APS
n=64

*
β2 GP1-D4/5

2 000
1 500
1 000
0 500
0 000
1-year-old
childrenn=57

ADchildren
n=33

APS
n=64

1-year-old
childrenn=57

ADchildren
n=33

APS
n=64

1-year-old
childrenn=57

Adapted from Andreoli, et al. Ann Rheum Dis (2010)

1-year-old children: one year-old children born to mothers with systemic autoimmune diseases; AD children: children
with atopic dermatitis; APS: patients with antiphospholipid syndrome.

Antiphospholipid Syndrome
Characteristics of criteria and non-criteria tests
According to the international consensus guidelines, a patient can be classified as having definite antiphospholipid
syndrome, if one of the clinical criteria and one of the laboratory criteria listed below are present:

Clinical Criteria
1. Vascular thrombosis: ≥1 arterial, venous, or small vessel thrombosis
2. Pregnancy morbiditya. ≥1 fetal death (at or beyond the 10th week of gestation)b. ≥1 premature birth before the 34th week of gestation because ofeclampsia, severe preeclampsia, or placental insufficiencyc. ≥3 consecutive (pre) embryonic losses (before the 10th week of gestation)

Laboratory Criteria
1. Anticardiolipin antibody (IgG and/or IgM) in medium or high titer(i.e., >40 MPL/GPL, or above th 99th percentile), on two or more
occasions at least 12 weeks apart
2. Anti-β2-glycoprotein-I antibody (IgG and/or IgM) in medium or high titer (i.e., above
the 99th percentile) on two or more occasions at least 12 weeks apart
3. Lupus anticoagulant (LAC) positivity on two or more occasions at least
12 weeks apartChart adapted from Miyakis M. International consensus statement on an update of the classification criteria for definite APS. Intl Soc Thromb and Haemos. 2006. 4: 295-306.

Criteria tests
aCL IgG and IgM• The aCL test is positive in more than 80-90% of patients with APS. However, these tests combined (IgG &IgM) offer modest specificity as this test may be positive in a variety of other disorders, including connective
tissue diseases and infectious diseases such as syphilis.15• Association of a positive aCL test with clinical manifestations of APS occurs principally with persistentmedium to high levels of aCL antibodies. IgG is more prevalent than IgM and IgA.

β2GP1 IgG and IgM• The β2GP1 test is more specific than aCL and should be used in conjunction with aCL tests.
• Omission of the β2GP1 test can result in failure to correctly classify as much as 30% of APS patients.17

LAC• The LAC test measures the ability of antiphospholipid antibodies to prolong phospholipid-dependent
clotting reactions.
• The antiphospholipid antibodies detected in aCL and LAC are specific for phospholipids, phospholipidbinding proteins, or a complex of these molecules. These antibodies are heterogeneous; the two sets (aCL
and LAC) do not necessarily identify the same antibodies.
• This test is not recommended for use when patients are under anti-coagulation therapy as these drugs may
interfere with LAC test results.
• Due to complexity and varied protocols for LAC, this test may be subject to variability of results.18

Non-criteria tests
aCL IgA• Genetic and ethnic background can impact the role of testing for IgA aCL, for example African-Americans,
Afro-Caribbean, and Hispanic patients with SLE may have elevated levels of IgA aCL.16• IgA aCL was the only aCL isotype present in 82% of aCL-positive Afro-Caribbean patients.16

β2GP1 IgA

• Testing for IgA β2GP1 is recommended when other aPL assays are negative and APS is suspected.
• Positivity for IgA β2GP1 is more common in women who experience unexplained recurrent spontaneousabortion and unexplained fetal death even when test results for other isotypes and LAC are negative.16

PS/PT IgG and IgM• A systematic review of data from over 7000 patients shows that:- When PT binds with PS, a conformational change occurs in PT allowing clinically relevant epitopes to be
expressed; antibodies directed towards these epitopes are distinct from either PS or PT antibodies alone.20- Routine measurement of PS/PT, but not PT or PS, is useful in establishing the thrombotic risk of patients.20

β2GP1 Domain 1• Domain 1 of β2GP1, the immunodominant epitope in APS, becomes exposed when bound to negatively
charged phospholipids.
• Test results for Domain 1 of β2GP1 may help guide clinical decision making given the marker's strong
association with increased risk of thrombosis.
• High titer anti-β2GP1 domain 1 antibodies are associated with a greater risk of thrombosis.7

Comprehensive offering of assays
MethodName

MethodName

QUANTA LiteELISA

aCL IgG
aCL IgM
aCL IgAaCL Screen
β2GP1 IgG
β2GP1 IgM
β2GP1 IgA
β2GP1 Screen
PS/PT IgG
PS/PT IgM
Phosphatidylserine IgG
Phosphatidylserine IgM
Phosphatidylserine IgA

QUANTA FlashCIA

aCL IgG
aCL IgM
aCL IgA
β2GP1 IgG
β2GP1 IgM
β2GP1 IgA
β2GP1 Domain 1

For more information contact your Inova representative or call customer service
at +1-858-586-9900, or visit our website at www.inovadx.com

References
1. Pengo V, Ruffatti A, Del Ross T, Tonello M, Cuffaro S, Hoxha A, Banzato A, Bison E, Denas G, Bracco A, Padayattil
Jose S. Confirmation of the initial antiphospholipid antibody positivity depends on antiphospholipid
antibody profile. J Thromb Haemost. 2013 Apr; 11(8):1527-31.
2. Sciascia S, Murru V, Sanna G, Roccatello D, Khamashta MA, Bertolaccini ML. Clinical accuracy for diagnosisof antiphospholipid syndrome in systemic lupus erythematosus: evaluation of 23 possible combinations ofantiphospholipid antibody specificities. J Thromb Haemost. 2012 Dec;10(12):2512-8.
3. Otomo K, Atsumi T, Amengual O, Fujieda Y, Kato M, Oku K, Horita T, Yasuda S, Koike T. Efficacy of the
antiphospholipid score for the diagnosis of antiphospholipid syndrome and its predictive value forthrombotic events.Arthritis Rheum. 2012 Feb;64(2):504-12.
4. Roubey RA. Risky business: the interpretation, use, and abuse of antiphospholipid antibody tests in clinicalpractice. Lupus. 2010 Apr;19(4):440-5.
5. Andreoli L, Tincani A. Beyond the "syndrome": antiphospholipid antibodies as risk factors. Arthritis Rheum.
2012 Feb;64(2):342-5.
6. Tarr T, Lakos G, Bhattoa HP, Shoenfeld Y, Szegedi G, Kiss E. Analysis of risk factors for the development ofthrombotic complications in antiphospholipid antibody positive lupus patients. Lupus. 2007;16(1):39-45.
7. Mahler M, Norman GL, Meroni PL, Khamashta, M. Autoantibodies to domain 1 of beta 2 glycoprotein 1: apromising candidate biomarker for risk management in antiphospholipid syndrome. Autoimmun Rev. 2012
Dec; 12(2):313-317.
8. V. Pengo, A. Ruffatti, M. Tonello, S. Cuffaro, A. Banzato, E. Bison, G. Denas, S. Padayattil Jose. Antiphospholipid
syndrome: Antibodies to Domain 1 of β2-glycoprotein 1 correctly classify patients at risk. J Thromb Haemost
2015
9. Meijide H, Sciascia S, Sanna G, Khamashta MA, Bertolaccini ML. The clinical relevance of IgA anticardiolipin
and IgA anti-β2 glycoprotein I antiphospholipid antibodies: a systematic review. Autoimmun Rev. 2013
Jan;12(3):421-5.
10. Pregnolato F, Chighizola CB, Encabo S, Shums Z, Norman GL, Tripodi A,Chantarangkul V, Bertero T, De Micheli
V, Borghi MO, Meroni PL. Anti-phosphatidylserine/prothrombin antibodies: an additional diagnostic marker
for APS? Immunol Res. 2013 Jul; 56(2-3):432-8.

11. Nayfe R, Uthman I, Aoun J, Saad Aldin E, Merashli M, Khamashta MA. Seronegative antiphospholipid
syndrome. Rheumatology (Oxford). 2013
12. Rodriguez-Garcia JL, Bertolaccini ML, Cuadrado MJ, Sanna G, Ateka-Barrutia O, Khamashta MA. Clinicalmanifestations of antiphospholipid syndrome (APS) with and without antiphospholipid antibodies (theso-called 'seronegative APS'). Ann Rheum Dis. 2012 Feb;71(2):242-4.
13. Kumar S, Papalardo E, Sunkureddi P, Najam S, González EB, Pierangeli SS. Isolated elevation of IgAanti-beta2glycoprotein I antibodies with manifestations of antiphospholipid syndrome: a case series of fivepatients. Lupus. 2009 Oct;18(11):1011-4.
14. Mehrani T, Petri M. Association of IgA Anti-beta2 glycoprotein I with clinical and laboratory manifestations ofsystemic lupus erythematosus. J Rheumatol. 2011; 38(3):450-3.
15. Amengual O, Atsumi T, Khamashta M, Hughes, G. Advances in APS. Ann Acad Med Singapore. 1998;27:61-66.
16. Bertolaccini M, Amengual O, Atsumi T, Binder W, et al. Non-criteria aPL tests: report of a task force andworkshop at the 13th International Congress on aPL. Lupus 2011;20:191-205
17. Nash M, Camilleri R, Kunka S, et al. The anticardiolipin assay is required for sensitive screening of aPL. J
Thromb Haemost. 2004; 2:1077-1081.
18. Pengo V, Tripodi A, Reber G, Rand JH, Ortel TL, Galli M, De Groot, PG. Update of the guidelines for lupus
anticoagulant detection. J Thromb Haemost 2009 7:1737-1740.
19. Vlagea A, Gil A, Cuesta MV, Arribas F, Diez J, Lavilla P, Pascual-Salcedo D. aPS/PT as potential markers of APS.
Clin Appl Thromb Hemost 2012 doi: 10.117/1076029612437578
20. Scascia S, Sanna G, Murru V, Roccatello D, Khamashta MA, Bertolaccini, ML. aPT and aPS/PT antibodies andthe risk of thrombosis in the APS. J Thromb Haemost. 2014; 111: 354-364.

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QUANTA Lite and QUANTA Flash are registered trademarks of Inova Diagnostics, Inc. BIO-FLASH is a registered trademark of Biokit S.A.
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