The detection of anti-β2GPI and anti-cardiolipin antibodies for the diagnosis of the antiphospholipid syndrome (APS) is mainly done by quantitative endpoint assays. Taking into account that not all antibodies directed against β2GPI and cardiolipin/β2GPI are pathogenic, such non-functional assays will result in a relatively high false positivity. Therefore, we believe the optimization of a functional assay for the diagnosis of APS and the prediction of thrombosis is warranted. Currently, the only functional assay included in the APS criteria is the lupus anticoagulant (LAC) assay. LAC assays rely on the prolongation of the clotting time through interaction of the autoantibody with phospholipids. However, LAC tests require an adequate plasma preparation and are complicated and labor-intensive. Additionally, specificity and sensitivity largely depend on the calculation of cut-off values, the choice of the reagents and a number of uncertainties exist in the interpretation of the results. The relevance of a weak LAC, and the influence of oral anticoagulants need to be further addressed.

A simple modification of the LAC test, in which cardiolipin was used as a confirmation reagent, enabled the discrimination between anti-β2GPI- and anti-prothrombin-dependent LAC, and the resulting assay proved to better correlate with thrombosis compared to the classic LAC. Furthermore, thrombin generation-based assays proved to be promising for the diagnosis of APS. Finally, the sensitivity of the activated protein C (APC) system has been found to be diminished in APS patients, as patients show a decreased response to both the addition of APC and thrombomodulin (TM). We recently combined these findings in one functional thrombin generation-based assay performed in the absence and presence of cardiolipin as well as thrombomodulin. Technical validation of the test is currently ongoing. As recommended by the SSC, cut-off values for this assay will be determined as the 99th percentile of a separate group of 120 healthy controls. A first clinical validation of the assay includes the measurement of normal pooled plasma (NPP) versus NPP supplemented with different monoclonal anti-β2GPI antibodies or anti-prothrombin antibodies isolated from APS patients.

Consequently, validation of our assay will be performed by testing samples of a multi-centre cohort we prepared in collaboration with Dr. Katrien Devreese (University of Ghent, Belgium). This cohort contains patient samples from 8 European centers, consisting of 259 thrombotic APS patients, 204 patients with a history of thrombosis and negative for laboratory criteria of APS, 122 obstetric APS patients, 33 patients with pregnancy complications and negative for laboratory criteria of APS, 196 patients with an autoimmune disease other than APS, 100 individuals with a normal pregnancy, 194 controls that were referred for antiphospholipid antibody testing for other reasons than the clinical criteria of APS and 60 women that were diagnosed with APS without specification of the clinical manifestations.

The development of this functional assay based on thrombin generation will hopefully result in a reduced false-positive and false-negative rate, as well as a better thrombotic risk stratification of the patients and consequently lead to an improved treatment strategy in APS.


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