A new functional prothrombin-based activated protein C (APC) resistance (APC-R) test (Pefakit® APC-R Factor V Leiden, Pentapharm, Basel, Switzerland) is presented.

Methods: The plasma sample is mixed with a reagent containing APC and snake venom specifically activating FV (RVV-V, Daboia russelli) and plasma that has been depleted of FV. During an incubation period of 180 sec the activated FV is inactivated by APC. Subsequently a reagent that contains a FV dependent prothrombin activator (Noscarin, Notechis scutatus) and EDTA is added. The clotting time is recorded. A second determination is performed under identical conditions, with the exception that no APC is added to the first reagent. A ratio between the two measurements is calculated. 703 samples of patients undergoing thrombophilia screening were analysed. Results were correlated to PCR based FVL testing, aPTT, PT, and to levels of Protein C, Protein S, Fibrinogen, FVIII and lupus anticoagulant index.

Results: Using a predefined cut-off of a ratio of 2.5 a 100% sensitivity and specificity for the detection of a FVL mutation was found. Using a cut-off ratio of 1.2 a complete but narrow distinction of FVL heterozygous (n=192) and FVL homozygous samples (n=27) was determined. No interference by sample’s INR and aPTT, PS, fibrinogen and FVIII levels and lupus anticoagulant ratio was detected.

Conclusion: The new snake-venom-based APC-R assay provides an improved distinction of FV wild-type and FVL carriers compared to the data reported for the aPTT based methods. The use of a FV dependent prothrombin activator eliminates effects of FVIII concentration or lupus anticoagulants in the sample.

Topics:
activated protein c resistance, factor v leiden, snakes, venoms, activated partial thromboplastin time measurement, antiphospholipid syndrome, prothrombin, fibrinogen, activated protein c, edetate disodium

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2005, The American Society of Hematology
2004
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