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Testing for Activated Protein C Resistance
Activated protein C (APC) resistance has a reported prevalence of 10%–15% and is a common cause of thrombophilia. The majority of APC resistance is directly attributable to heritable mutations in coagulation factor V (FV), primarily the FV Leiden (FVL) R506Q mutation, which results in decreased efficiency of FVa inactivation by APC due to alteration of one of the APC cleavage sites. However, other less common mutations in FV have also been associated with in vitro APC resistance and have variable associations with thrombosis. APC resistance that is independent of FVL has also been reported and can be attributable to increased levels of factor VIII or prothrombin, presence of a lupus anticoagulant, oral contraceptive use, or pregnancy.
Activated Protein C Resistance Assays
APC resistance assays are fluid-phase functional assays that measure the ability of protein C to inactivate FVa and FVIIIa. The primary indication for this assay is the clinical assessment of thrombophilia.
Methods
Most commercially available assays for the detection of APC resistance utilize activated partial thromboplastin time (APTT)–based or prothrombin time (PT)–based assays for detection. However, Russell viper venom–based assays with or without the addition of Noscarin, a snake venom that activates prothrombin in a FVa-dependent manner, assays utilizing the endogenous thrombin potential, and an assay utilizing a diluted plasma supplemented with APC and nonactivated clotting factors triggered with purified Xa, phospholipids, and calcium have also been employed for this purpose. Regardless of the activator, the principle of these assays involves measuring a clotting time; i.e., APTT before and after the addition of APC and calcium to a plasma sample. Theoretically, the addition of APC to a normal plasma sample should prolong the clotting time (e.g., APTT) because of inactivation of FVa and FVIIIa, whereas in an APC-resistant individual the degree of prolongation of the clotting time (e.g., APTT) should be decreased. As a result, a calculation known as the APC sensitivity ratio is performed:
A resultant ratio less than or equal to the cutoff value is consistent with resistance to APC. For instance, it has been reported that most patients with ratios less than 0.71 using an APTT-based assay are heterozygous or homozygous for FVL.
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