Interference in protein S activity testing and free protein S: impact of lupus anticoagulant, elevated factor VIII,...

Protein S (PS), a key regulator in coagulation and inflammation, is typically measured via clot-based assays prone to interference from lupus anticoagulant (LA), elevated Factor VIII (FVIII), and anticoagulants, potentially leading to misinterpretation. Free PS (FPS), measured by immunoturbidimetry, is less affected by such interferents. This study evaluates how these variables influence PS activity and FPS levels.

We analyzed plasma from 263 patients, including 54 tested for FVIII activity, 34 LA-negative controls without anticoagulation, 58 patients testing LA-positive and 37 on warfarin. An ex vivo experiment was conducted using heparin, argatroban, bivalirudin and rivaroxaban addition experiment was performed using plasma from 80 healthy, LA-negative individuals not receiving anticoagulants. PS activity and FPS were measured in all samples, followed by statistical analysis.

LA positivity was associated with increased measured PS activity and FPS levels. In patients with elevated FVIII, PS activity decreased while FPS levels increased. Warfarin therapy caused a decrease in measured PS activity, while the addition of argatroban, bivalirudin, and rivaroxaban led to a concentration-dependent pseudo-increase in PS activity. FPS levels remained unaffected by all anticoagulants tested.

LA positivity is associated with artificially increased PS activity, whereas elevated FVIII is associated with reduced measured PS activity. Therefore, reduced PS activity observed under these conditions should be interpreted with caution, as it may reflect assay interference rather than true PS deficiency. Direct thrombin inhibitors and rivaroxaban also cause significant overestimation of PS function. Measurement of FPS provides a robust and complementary tool to mitigate the risk of misdiagnosing PS deficiency in the presence of these common interferents.