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

Clotting-based activated protein C (APC) assays have limitations when testi ng patients on oral anticoagulant (OA) therapy or with a lupus anticoagulan t. (LA). Predilution in factor V (FV)-deficient plasma and testing with pho spholipid-rich Russell Viper venom (RVV)-based methods have been shown to b e the most suitable methods when testing these patient groups, respectively . We evaluated a modified RVV based clotting test (Gradileiden V test; Grad ipore, Sydney, Australia) in a large patient cohort and determined its sens itivity to the FV Leiden mutation. We also examined whether normal plasma c an be used to dilute plasma from warfarinized patients without compromising sensitivity to the EV Leiden mutation. A total of 1956 plasmas were studie d including congenital protein C (five plasmas), and protein S deficiency ( five plasmas), LA (29 plasmas), FV Leiden heterozygote (102 plasmas), and h omozygote (five plasmas), warfarin (54 plasmas), standard heparin therapy ( 37 plasmas) and normal healthy controls (21 plasmas). Molecular analysis wa s performed on all samples. The effect of EV Leiden concentration on the AP C ratio was examined by determining the APC resistance of a homozygous plas ma 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 dilu tions of 1/4 (25% FV Leiden) to 1/32 (3% FV Leiden). A 1/2 dilution in pool ed NP is recommended for patients on OA therapy because the test remains se nsitive 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 t he APC ratio was similarly studied by determining the APC resistance of a h omozygous plasma serially diluted in two sources of LA-positive plasma. Thi s relationship was also non-linear and dependent on the initial APC ratio o f 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 o f 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 pati ents with a LA and patients receiving warfarin providing a 1/2 predilution step in pooled NP is performed, fooled NP does not affect the sensitivity o f the test to the mutation, is routinely used in coagulation laboratories, and is considerably less expensive than FV-deficient plasma. (C) 2000 Lippi ncott Williams & Wilkins.