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Measurement of Factor V Activity in Human Plasma Using a Microplate Coagulation Assay
Published on: September 9, 2012
Modified APC-resistance test: variable ratios with respect to source of factor V-deficient plasma
C Ts'ao1, D Neofotistos, M Oropeza
1Department of Pathology, Northwestern Memorial Hospital, Chicago, Illinois 60611, USA.
Insights
A modified assay using factor V (FV)-deficient plasma can screen for APC-resistance, a thrombosis risk factor, in patients on warfarin. However, results differ from the original method and vary with FV-deficient plasma source.
Area of Science:
- Hematology
- Thrombosis Research
- Clinical Chemistry
Background:
- Factor V Leiden (FV-Leiden) mutation causes APC-resistance, a major venous thrombosis risk factor in Caucasians.
- The activated partial thromboplastin time (APTT) based APC ratio is a common screening method.
- Standard APC ratio testing is unreliable in patients on oral anticoagulants due to vitamin K-dependent clotting factor interference.
Purpose of the Study:
- To evaluate a modified APC-sensitivity assay using FV-deficient plasma for screening APC-resistance.
- To assess the assay's applicability in patients on warfarin therapy.
- To investigate the impact of different commercial FV-deficient plasma sources on APC ratio results.
Main Methods:
- A modified APC-sensitivity assay was developed, involving dilution of test plasma with FV-deficient plasma.
- The modified assay was tested on normal individuals, FV-Leiden positive individuals, and patients on warfarin.
- APC ratios from the modified assay were compared to those from the original method, using FV-deficient plasma from various commercial sources.
Main Results:
- APC ratios determined by the modified method differed significantly from the original method across all tested groups.
- The modified method successfully identified APC-resistance in patients on warfarin therapy.
- Significant variations in APC ratios were observed depending on the commercial source of the FV-deficient plasma used.
Conclusions:
- The modified APC-sensitivity assay using FV-deficient plasma is a viable method for detecting APC-resistance in patients on warfarin and other individuals.
- Clinicians must be aware that APC ratios obtained with the modified method are not directly comparable to the original method's results.
- The choice of commercial FV-deficient plasma source can influence APC ratio outcomes in the modified assay, necessitating careful consideration and standardization.
Abstract:
A single point mutation of the factor V (FV) gene, leading to the substitution Arg506Gln in the FV molecule (FV-Leiden) and hence resistance to its breakdown by activated protein C (APC), is the most prevalent risk factor for venous thrombosis in the Caucasians. A ratio determined by activated partial thromboplastin time (APTT) of test plasma in the presence or absence of exogenous APC (the APC ratio), is the method widely used to screen individuals with this risk factor for thrombosis. Because of functional defects of vitamin K-dependent clotting factors in patients on oral anticoagulant therapy, this method cannot be applied to those patients without modification. One modification is to mix test plasma (1:5 or 1:10) with FV-deficient plasma so that 80-90% of functioning vitamin K-dependent factors are supplied by the FV-deficient plasma. Even with 10-20% of FV in the mixture, APC-resistance still can be demonstrated. In this report, we present our results of the modified APC-sensitivity assay using FV-deficient plasma from different commercial sources. APC ratios determined by the original method in which test plasma is not mixed with FV-deficient plasma can be significantly different from those determined by the modified method in which test plasma is diluted 1:5 with FV-deficient plasma. This difference between methods was observed not only in normal individuals, but also in FV-Leiden positive individuals, and in patients on warfarin therapy. Further, APC ratios varied significantly depending on the commercial source of the FV-deficient plasma. The modified method is apparently suitable to identify APC-resistance in patients on warfarin therapy, as well as in individuals not receiving anticoagulant treatment. However, one must be aware that APC-resistance ratios obtained with the modified method are likely to be different from those established with the original method, and the source of FV-deficient plasma can be a factor influencing the ratios in the former cases.
