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.
1. Devreese K, Hoylaerts MF. Laboratory diagnosis of the antiphospholipid syndrome: a plethora of obstacles to overcome. Eur J Haematol. 2009;83(1):1-16 2. De Laat B, Derksen RH, Reber G, Musial J, Swadzba J, Bozic B, Cucnik S, Regnault V, Forastiero R, Woodhams BJ, De Groot PG. An international multicentre-laboratory evaluation of a new assay to detect specifically lupus anticoagulants dependent on the presence of anti-beta2-glycoprotein autoantibodies. J Thromb Haemost. 2011;9(1):149-53 3. Dargaud Y, Trzeciak MC, Bordet JC, Ninet J, Negrier C. Use of calibrated automated thrombinography +/- thrombomodulin to recognise the prothrombotic phenotype. Thromb Haemost. 2006;96(5):562-567. 4. Devreese K, Peerlinck K, Arnout J, Hoylaerts MF. Laboratory detection of the antiphospholipid syndrome via calibrated automated thrombography. Thromb Haemost. 2009;101(1):185-96 5. Zuily S, Ait Aissa K, Membre A, Regnault V, Lecompte T, Wahl D. Thrombin generation in antiphospholipid syndrome. Lupus. 2012;21(7):758-760. 6. Liestol S, Sandset PM, Mowinckel MC, Wisloff F. Activated protein C resistance determined with a thrombin generation-based test is associated with thrombotic events in patients with lupus anticoagulants. J Thromb Haemost. 2007;5(11):2204-2210.