Clinical Evaluation of a New Functional, Plasma-Based Assay for the Detection of Factor V Leiden - the Pefakit® APC-R Factor V Leiden Assay.
Authors: Hoppensteadt Debra A. , Cunanan Josephine , Walenga Jeanine M. , Fareed Jawed , Wilmer Marianne , Janssen Michael 
Published: October 2019

Abstract
Activated protein C (APC) resistance is the most frequent hereditary defect associated with deep vein thrombosis (DVT). Over 80% of the APC resistance phenotypes can be explained by the Factor V Leiden (FVL) mutation. This defect is caused by a point mutation in the factor V gene resulting in a replacement of the amino acid Arg 506 by a Gln residue. For patients who are at risk of thrombosis or who have active thrombosis, it is of interest to be able to screen for the FVL mutation in a easy and reliable manner. There are two types of assays for the detection of FVL. The genotype is reliably detected by PCR technology, but this technology is not readily available to all laboratories nor is it inexpensive. The phenotypic expressions of the defect can be identified by plasma-based functional assays, of which several are commercially available. The functional Pefakit® APC-R FVL assay (Pentapharm; Basel, Switzerland) (APC-R) is a new plasma-based clotting assay developed to overcome the limitations of the current assays, including sensitivity and specificity. Additionally, the APC-R assay is more informative as results specify heterozygous / homozygous opposed to a report of normal / abnormal by all other assays. The APC-R assay is designed to specifically act at the level of the prothrombinase complex using a factor V dependent prothrombin activator isolated from the snake venom of the Notechis scutatus. We investigated the phenotypic and genotypic expressions of FVL in patients with cancer and acute symptomatic DVT and/or PE (n=67). Normal healthy volunteers (n=50) collected in-house served as controls. DNA isolated from buffy coats obtained from citrated anticoagulated blood was used to profile FVL using standard PCR probes. Citrated plasma was used to determine APC-R by the functional Pefakit® assay. Of the 67 patients profiled by molecular analysis, 5 (7%) were heterozygous and 1 (1%) was homozygous for the FVL defect. In the normal healthy volunteers, 3 (6%) were heterozygous and 1 (2%) was homozygous for FVL. Using the APC-R functional plasma-based assay, 4 cancer patients (6%) were heterozygous and 2 (3%) were homozygous. In the normal volunteers, 4 (8%) were heterozygous and 1 (2%). was homozygous with the APC-R assay. The APC-R functional plasma-based assay did not miss any FVL patient shown positive by PCR. The heterozygous cancer patients detected with the functional assay were detected as heterozygous with the PRC method; one patient detected as homozygous with the functional method was determined to be heterozygous by PCR. In the normal healthy volunteers, 1 volunteer that was heterozygous by the plasma based assay was negative by PCR. This study demonstrates that the prevalence of the FVL defect in cancer patients with thrombosis is similar to the normal healthy population. Although additional studies to validate the sensitivity/specificity of the APC-R assay and to establish its positive / negative predictive values are needed, the results of this first investigation suggest that the APC-R assay may provide more useful results than other commercially available assays for the detection of FVL. The results of this study showed a good correlation of the functional FVL. APC-R assay with the reference standard molecular PCR method. These data suggest a practical role of this new assay in the clinical laboratory diagnosis of FVL.