Diagnostic Criteria for Antiphospholipid Antibody Syndrome

Laboratory Criteria for APS

The diagnosis of Antiphospholipid Syndrome (APS) requires both clinical criteria (thrombosis or pregnancy morbidity) and laboratory criteria, with positive laboratory tests confirmed after 12 weeks, as recommended by the Scientific Standardization Subcommittee (SSC) on Lupus Anticoagulant/Phospholipid Antibodies 1
Lupus anticoagulant (LAC) present in plasma is a well-established thrombotic risk factor, particularly when associated with other antiphospholipid antibodies, and requires a combination of two phospholipid-dependent clotting assays for detection 1, 2
β2GPI-dependent anticardiolipin antibodies (aCL) of IgG/IgM isotype in plasma or serum should be present at levels higher than the 99th percentile of normal controls, measured by solid phase assays (ELISA or automated systems), according to the SSC on Lupus Anticoagulant/Phospholipid Antibodies recommendations 2
Anti-β2-glycoprotein I antibodies (aβ2GPI) of IgG/IgM isotype in plasma or serum should be present at levels higher than the 99th percentile, measured by solid phase assays (ELISA or automated systems) 2

Positive laboratory tests must be confirmed after at least 12 weeks to avoid over-diagnosis from transient antibody positivity, as recommended by the SSC on Lupus Anticoagulant/Phospholipid Antibodies 1, 3
All three tests (LAC, aCL, and aβ2GPI) should be performed, preferably on the same sample, to characterize the patient's antibody profile, with triple positivity (LAC + aCL + aβ2GPI of same isotype) showing the strongest association with thrombotic and obstetric APS 4, 5
IgG and IgM isotypes are included in the classification criteria, while the significance of IgA aCL and aβ2GPI remains controversial and requires further research 1, 6, 7

β2GPI domain I antibodies (aD1) are a subgroup of IgG aβ2GPI strongly associated with thrombosis, mainly present in triple-positive patients, and currently considered a confirmation of higher thrombotic risk rather than a replacement for aβ2GPI testing 3, 5
Antibodies to phosphatidylserine/prothrombin (aPS/PT) show potential diagnostic value, mainly associated with LAC, but additional value in APS diagnosis needs further confirmation 3

Test results should always be related to clinical symptoms, requiring interaction between laboratory and clinician, with awareness of inter-assay and inter-laboratory variability 4
For clinical diagnosis, all test results above the cut-off value (>99th percentile) should be regarded as positive, with triple-positive patients indicating high risk of recurrence of thrombosis or development of a first thrombosis 4, 5, 6
The 2023 ACR/EULAR APS classification criteria maintain the same laboratory parameters but have abandoned the 99th percentile cutoff in favor of moderate (40 Units) and high (80 Units) titer thresholds, as recommended by the American College of Rheumatology (ACR) and the European League Against Rheumatism (EULAR) 8

The International Society on Thrombosis and Haemostasis (ISTH) guidelines state that repeat testing after 3 months is designed to avoid over-diagnosis by distinguishing persistent from transient positivity of antibodies, and this requirement applies exclusively to positive test results 9, 10, 11.
The 12-week confirmation requirement for positive test results ensures reliability and persistence of antibody positivity before making an APS diagnosis, as recommended by the ISTH 12, 13.

If strong clinical suspicion persists (recurrent thrombosis, pregnancy morbidity) with negative standard aPL testing, consider retesting with alternative methods or at a later time point, according to the ISTH guidelines 9, 10.
Repeat testing should be performed if initial negative results were obtained during anticoagulation therapy, acute thrombosis, or pregnancy, as these conditions can produce false negative results, as stated by the ISTH 12, 14.
Factor VIII increases during pregnancy can mask lupus anticoagulant by shortening APTT, and antibody levels may decrease during acute thrombosis due to antibody deposition at the thrombotic site, as noted by the ISTH 12.
Anticoagulants (VKA, DOAC, heparin) can interfere with LA testing, producing unreliable results, according to the ISTH 12, 13, 14.

Low positive or equivocal results near the cutoff value should be repeated to confirm true positivity or negativity, as assay imprecision (up to 10%) can affect classification around cutoff values, as stated by the ISTH 12.
Test results must always be interpreted in clinical context with close laboratory-clinician interaction, as recommended by the ISTH 9, 10, 13.

The International Society on Thrombosis and Haemostasis (ISTH) specifies that the 12‑week confirmation rule is intended to differentiate persistent from transient lupus anticoagulant (LA) positivity and applies only to positive test results【15】.
Laboratory criteria for antiphospholipid syndrome (APS) require two positive LA tests spaced at least 12 weeks apart; an initial positive result followed by a negative result at 8 weeks does not satisfy these criteria【15】【16】【17】.
Consequently, when the first LA test is positive and the second test at 8 weeks is negative, no 12‑week repeat testing is required, and APS cannot be diagnosed on this basis【15】.

A positive LA result that becomes negative at 8 weeks reflects transient antibody positivity, not persistent disease【15】.
Transient LA positivity is common in the setting of infections, certain drugs, or acute illnesses, which is why the ISTH confirmation requirement targets only persistent positives【15】【16】.
Interfering conditions (e.g., ongoing anticoagulation therapy with vitamin‑K antagonists, direct oral anticoagulants, heparin; acute thrombosis; pregnancy) can produce false‑negative LA results; retesting after resolution of these factors is advisable【15】.

During pregnancy, elevated factor VIII shortens the activated partial thromboplastin time (aPTT) and can mask LA detection; approximately 25 % of pregnant patients who were LA‑positive earlier become negative in the second or third trimester【15】.

Around the time of an acute thrombotic event, circulating LA antibodies may decrease due to deposition at the clot site, with levels potentially rising again after the acute phase【15】.

If the initial positive LA was obtained during an acute illness, infection, or while the patient was on medications known to cause transient positivity, the subsequent negative result likely represents resolution of a transient antibody rather than a testing error【15】.
No routine schedule exists for retesting a negative LA result; repeat testing should be considered only when clinical circumstances change (e.g., resolution of interfering factors or persistent high clinical suspicion)【15】.
In cases of high clinical suspicion for APS (e.g., recurrent thrombosis, obstetric morbidity) despite a negative 8‑week test, clinicians should discuss the uncertainty with the patient and may repeat testing at a later, unspecified time point after interfering factors have been addressed【15】.