Determination of activated protein C resistance in anticoagulated and lupus positive patients

Abstract

Clotting-based activated protein C (APC) assays have limitations when testing patients on oral anticoagulant (OA) therapy or with a lupus anticoagulant (LA). Predilution in factor V (FV)-deficient plasma and testing with phospholipid-rich Russell Viper venom (RVV)-based methods have been shown to be the most suitable methods when testing these patient groups, respectively. We evaluated a modified RVV based clotting test (Gradileiden V test; Gradipore, Sydney, Australia) in a large patient cohort and determined its sensitivity to the FV Leiden mutation. We also examined whether normal plasma can be used to dilute plasma from warfarinized patients without compromising sensitivity to the FV Leiden mutation. A total of 1,956 plasmas were studied including congenital protein C (five plasmas), and protein S deficiency (five plasmas), LA (29 plasmas), FV Leiden heterozygote (102 plasmas), and homozygote (five plasmas), warfarin (54 plasmas), standard heparin therapy (37 plasmas) and normal healthy controls (21 plasmas). Molecular analysis was performed on all samples. The effect of FV Leiden concentration on the APC ratio was examined by determining the APC resistance of a homozygous plasma serially diluted in six sources of normal plasma (NP). The relationship was non-linear and dependent on the initial APC ratio of the chosen source of NP. APC resistance was demonstrated in the varying sources of NP in dilutions of 1/4 (25% FV Leiden) to 1/32 (3% FV Leiden). A 1/2 dilution in pooled NP is recommended for patients on OA therapy because the test remains sensitive at levels of 25% FV Leiden and this is the dilution routinely used for other applications in a coagulation laboratory. The effect of a LA on the APC ratio was similarly studied by determining the APC resistance of a homozygous plasma serially diluted in two sources of LA-positive plasma. This relationship was also non-linear and dependent on the initial APC ratio of the LA-positive plasma. APC resistance was demonstrated in dilutions of 1/16 (6% FV Leiden) to 1/64 (1.5% FV Leiden) demonstrating the sensitivity of the test to APC resistance in the presence of a LA. Our results show the modified RVV-based test clearly predicts the presence of factor V Leiden in a large cohort of patients. The method offers advantages when testing patients with a LA and patients receiving warfarin providing a 1/2 predilution step in pooled NP is performed. Pooled NP does not affect the sensitivity of the test to the mutation, is routinely used in coagulation laboratories, and is considerably less expensive than FV-deficient plasma.