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Measurement of Factor V Activity in Human Plasma Using a Microplate Coagulation Assay
Published on: September 9, 2012
Modification of the ProC Global assay using dilution of patient plasma in factor V-depleted plasma as a screening
J C Quincampoix1, M Legarff, C Rittling
1Laboratoire d'Hématologie, Hôpital Cochin, Paris, France.
Insights
A modified ProC Global assay effectively screens for factor V Leiden mutation, a common inherited risk for thrombosis. This enhanced assay demonstrates 100% sensitivity and specificity in identifying activated protein C resistance.
Area of Science:
- Hematology
- Clinical Diagnostics
- Molecular Genetics
Background:
- Factor V Leiden mutation is the most prevalent inherited risk factor for thrombosis in Caucasians.
- Laboratory diagnosis often relies on detecting poor anticoagulant response to activated protein C (APC).
- The ProC Global assay evaluates protein C pathway functionality by measuring APC's effect on activated partial thromboplastin time.
Purpose of the Study:
- To assess the performance of a modified ProC Global assay in screening for factor V Leiden mutation-related APC resistance.
- To evaluate a specific modification involving a 1:5 pre-dilution of patient plasma in factor V-depleted plasma.
Main Methods:
- Investigated 341 frozen plasma samples from patients with a history of venous thromboembolism.
- Employed a modified ProC Global assay with a 1:5 pre-dilution of patient plasma in factor V-depleted plasma.
- Analyzed sensitivity and specificity for factor V Leiden mutation detection.
Main Results:
- The modified assay achieved 100% sensitivity for the factor V Leiden mutation, correctly identifying all carriers (homozygotes and heterozygotes).
- Specificity was also 100%, with no false positives in patients with protein C deficiency, protein S deficiency, or other conditions.
- A normalized ratio below 0.80 indicated a decreased response, effectively flagging carriers of the mutation.
Conclusions:
- The modified ProC Global assay, using a 1:5 pre-dilution, shows high accuracy for screening factor V Leiden mutation-related APC resistance.
- This method is a potentially valuable screening tool for patients with a history of thrombosis.
- Further multicenter studies are recommended before widespread adoption, and laboratories should establish their own reference ranges and cut-off levels.
Abstract:
The activated protein C (APC) resistant-factor V (factor V Leiden) has emerged as the most common inherited risk factor for thrombosis in the Caucasian population. Beside DNA analysis, the laboratory diagnosis is often based on the detection of a poor anticoagulant response to exogenous APC. The ProC Global assay (Dade Behring, Marburg, Germany) is a global clotting assay, which was primarily developed to evaluate the functionality of the protein C anticoagulant pathway. It is based on the ability of endogenous APC, generated by activation of protein C by an extract from Agkistrodon contortrix contortrix venom, to prolong an activated partial thromboplastin time. It was previously found to be highly sensitive for the factor V Leiden mutation and for protein C deficiency, but only moderately sensitivity for protein S deficiency. Here, we evaluated the performance of a modification of the ProC Global assay using a 1 : 5 pre-dilution of patient plasma in factor V-depleted plasma in the screening of the factor V Leiden mutation-related APC resistance. For that purpose, we investigated selected frozen plasma samples from 341 patients with a history of venous thromboembolism. The sensitivity for the factor V Leiden mutation of the modified assay was found to be 100%, as all the carriers of that mutation (five homozygotes and 77 heterozygotes) had a decreased response to the assay, i.e. a normalized ratio below 0.80. Its specificity was also 100% since none of the other tested patients had a decreased response, i.e. isolated protein C (n = 3) or protein S deficiency (n = 50), or without any abnormality of the protein C pathway (n = 143), even those on oral anticoagulant treatment (n = 76). However, it would be preferable that each laboratory defines both its reference range and its cut-off level. Finally, even if larger-scale multicentre studies are needed before definite recommendations could be made, these results suggest that the ProC Global performed using a 1 : 5 pre-dilution of the patient plasma in factor V-depleted plasma could be validly used as a screening assay of the factor V Leiden mutation-related APC resistance in patients with a history of thrombosis.
