Activated Protein C resistance and plasma based functional clotting assays

  

 

Activated protein C (APC) resistance is the most frequent hereditary defect associated with deep vein thrombosis. Over 95% of the APC resistance phenotype can be explained by the Factor V Leiden mutation caused by an arginine to glycine point mutation at the 506 amino acid site in the factor V (FV) gene. The resulting Leiden variant of FV cannot be inactivated by activated protein C. The Factor V Leiden allele is present in about 5% of the Caucasian individuals and is virtually absent in Africans, Asians, and races with Asian ancestry. The heterozygous defect is associated with a 5 to 10 fold thrombosis risk and the homozygous defect is associated with a 50 to 100 fold increased thrombosis risk.

Different methods of detecting factor V Leiden include plasma based functional assays to identify phenotypic expression of the defect or genotype determination which can be done by PCR technology. Comparisons between the two assays indicate that there is good correlation between plasma based and DNA assays.

In plasma-based functional clotting assays, sample plasma is diluted with FV deficient plasma and incubated with FV activator from venom of Daboia russelli (the Russell viper, above) in order to convert FV into FVa. Coagulation is triggered by the addition of a FVa dependent prothrombin activator from snake venom from Notechis scutatus (the Tiger snake, above) in the absence of calcium. The clotting times are recorded and the ratios of clotting time (+APC/–APC) are calculated. Since the patient sample is diluted in FV deficient plasma, the assay can be run on patients on oral anticoagulant therapy and patients in the therapeutic range of heparin. Differentiation of homozygous, heterozygous, and negative samples is based on reference interval measurements of known genotyped patient plasmas and the cutoff ranges can vary depending on the instrument used. Although in these tests the data generally appears to have clean trimodal separation, consideration should also be given for indeterminate values which would indicate further studies such as genetic testing. 

Pefakit APC-R Factor V Leiden. Pentapharm package insert – for in-vitro diagnostic use. August 2009.

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