Clinical Information

Antiphospholipid syndrome (APS) has traditionally been described as a systemic autoimmune disease characterized by thrombosis and/or specific pregnancy-related morbidities associated with persistent documentation of "criterial" antiphospholipid antibody (aPL) tests.(1,2) Based on the 2006 revised Sapporo consensus classification, the "criterial" aPL antibody tests include lupus anticoagulant (LAC) and IgG/IgM antibodies to the cardiolipin (aCL) and beta2-glyocoprotein I (anti-B2GPI) with all tests carrying equal diagnostic significance for disease.(1) In 2023, the American College of Rheumatology (ACR)/ European League Against Rheumatism (EULAR) published new classification criteria for APS that includes an entry criterion of at least one positive aPL antibody test within 3 years of identification of an aPL-associated clinical criterion, followed by additive weighted criteria (score range 1-7 points each) clustered into 6 clinical domains (macrovascular venous thromboembolism, macrovascular arterial thrombosis, microvascular, obstetric, cardiac valve, and hematologic) and 2 laboratory domains (LAC functional coagulation assays, and solid-phase enzyme-linked immunosorbent assays (ELISA) for IgG/IgM aCL and/or IgG/IgM anti-B2GPI).(3)

Unlike LAC, which is evaluated using functional assays, diverse solid-phase immunoassays (SPA) such as ELISA, multiplex bead assay, chemiluminescent immunoassay, and fluorescence enzyme immunoassay are used in the clinical laboratories for the detection and measurement of aCL and anti-B2GPI IgA, IgG, and IgM antibodies.(4,5) For aCL IgG and IgM determinations, the APS classification guidance recommends antibody cut-off values greater than 40 IgG phospholipid (GPL) or IgM phospholipid (MPL) units (units traceable to the Harris standards for aCL antibody assays) or more than the 99th percentile for the testing laboratory's population for positivity. It also advocates for the use of values greater than the 99th percentile for the laboratory's population in the establishment of reference intervals for anti-B2GPI IgG and IgM antibody tests.(1) The use of cutoff values greater than 40 GPL or MPL units to define positivity is not be applicable to all aCL antibody immunoassays, as the threshold used to distinguish moderate-to-high positive from low positive results are test dependent.(5-7) In addition, the cutoff used at the 99th percentile of a laboratory's testing population may not be consistent with kits from the same manufacturer or 40 GPL units, in the case of aCL antibodies.(2,5-7)

Early observations that aCL antibody determinations made in the presence of B2GPI were more specific for APS led to the recommendation of B2GPI-dependent cardiolipin ELISA for APS evaluation.(1,8) Cardiolipin is a negatively charged phospholipid (PL) capable of binding diverse proteins, of which B2GP1 is one of the best characterized in APS. B2GP1 is a 326-amino acid protein that contains five repetitive structures or "sushi domains," termed domain 1 through 5, for a combined molecular weight of 54 kDa for the protein.(8) Anti-B2GP1 antibodies associated with thromboembolic events target domain 1 of the molecule and are responsible for LAC (functional, phospholipid-dependent prolongation of the clotting time) and aCL antibody positivity.(2) Compared to LAC and anti-B2GPI IgG antibodies, aCL IgG antibodies are less specific but sensitive for the diagnosis of APS. Of the aCL IgG and IgM, the IgG and not IgM confers higher diagnostic relevance and risk for definite APS.(1,3,6,7)

Thrombosis and obstetric complications are common clinical events in the general population and are not unique to APS; therefore, the presence of aPL antibodies is an absolute requirement for the diagnosis of definite APS.(1,2,6) Furthermore, aPL antibodies are heterogeneous with overlapping tendencies; the lack of aPL test harmonization or standardization requires the use of all three tests for optimal APS diagnosis.(1,3) The aPL antibodies were traditionally determined using classic ELISA, with more diverse methods recently developed and adapted for clinical testing. Recognizing the analytical and diagnostic challenges associated with aPL antibody testing, initiatives to support assay harmonization and utilization, including the development of calibrators, test development, and validation efforts as well as preanalytical, analytical, and postanalytical measures have been published. (2,4,5,7) Based on these and other published studies, the interpretation and relevance of aPL antibody tests are dependent on factors such as the type of aPL (LAC, aCL or anti-B2GPI), the source of cardiolipin and/or B2GPI, aPL antibody class (IgG, IgM, or IgA) and level as well as whether antibody positivity is single, double, or triple.(1,7)

The 2023 ACR/EULAR classification criteria for APS are meant for clinical studies and may not be appropriate for routine patient evaluation and management. Therefore, in clinical practice, if suspicion for disease is high but criteria aPL antibody tests are inconclusive or negative, deviation from the APS diagnostic criteria may be justified. This may include testing for noncriteria aPL antibody tests such the aCL IgA, anti-B2GPI IgA and anti-phosphatidylserine/prothrombin complex IgG and IgM antibodies.(2,5,9,10) However, there is no formal guidance for the measurement and interpretation of these noncriteria aPL antibodies in patients with APS or SLE.