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Diagnosis strategies in activated protein C resistance: is genotyping still necessary?
C Biron1, H Lamarti, J F Schved
1Laboratoire Central d'Hématologie, Hôpital Saint-Eloi, Montpellier, France.
Insights
A modified activated protein C (APC) resistance test effectively screens for Factor V Leiden mutation, reducing the need for genetic testing in most patients. However, genotyping remains crucial for abnormal results due to potential false positives.
Area of Science:
- Genetics
- Hematology
- Molecular Biology
Background:
- Activated protein C (APC) resistance is primarily caused by the Factor V Leiden (FVL) mutation.
- FVL is a major inherited risk factor for thrombosis, increasing laboratory diagnostic demand.
- Efficient screening strategies are crucial to manage increasing diagnostic workloads and associated costs.
Purpose of the Study:
- To develop and validate a cost-effective laboratory screening strategy for APC resistance.
- To compare a novel DNA assay (DGGE) with phenotypic assays for APC resistance diagnosis.
- To determine the utility of a modified functional APC resistance test in identifying FVL mutations.
Main Methods:
- Development of a DNA assay using denaturing gradient gel electrophoresis (DGGE).
- Systematic comparison of DNA analysis with phenotypic assays in 94 consecutive patients.
- Utilized a modified functional APC resistance test with a 1:5 plasma predilution and established cut-off values.
Main Results:
- A cut-off APC resistance ratio of ≤2.6 accurately identified individuals with normal phenotypes, yielding a predictive value of 1.0 for heterozygote FVL.
- A cut-off ratio of 1.5 effectively distinguished between homozygous and heterozygous FVL patients.
- False-positive results, such as lupus anticoagulant, necessitate genotyping for patients with abnormal test ratios.
Conclusions:
- The modified functional APC resistance test provides a reliable method for screening FVL, reducing the need for genotyping in individuals with normal phenotypes.
- Genotyping remains essential for confirming FVL in patients with abnormal APC resistance ratios due to potential interference from other conditions.
- The proposed strategy effectively lowers the number of samples requiring expensive DGGE analysis.
Abstract:
Resistance to activated protein C (APC) is due, in most cases, to a G to A mutation at nucleotide 1691 of factor V (FV) gene (the Leiden mutation). This inherited abnormality is now considered to be the major hereditary cause associated with an elevated risk of thrombosis. For this reason, laboratories are faced with an increasing number of samples referred for APC resistance diagnosis. This could have serious economic consequences and a comprehensive laboratory screening strategy for APC resistance is necessary. An original DNA assay based on denaturing gradient gel electrophoresis (DGGE) was designed in our laboratory. During a first period we systematically performed DNA analysis and compared the results with phenotypic assays. Using the modified functional test with a 1:5 predilution of plasmas, the cut-off value for APC resistance ratio was 2.6 in our sample. Among 94 consecutive patients referred to our laboratory we found a clear cut-off between the APC resistance ratio obtained for normal and abnormal individuals. The modified test had a predictive value of 1.0 found by a cut-off < or = 2.6 for the heterozygote FV Leiden. This obviates the necessity of genotyping subjects with a normal phenotype. Among patients with an abnormal phenotype we were able to fully discriminate between homozygous and heterozygous patients using a cut-off value of 1.5. Nevertheless, our results demonstrate that, because of false-positive results such as lupus anticoagulant, genotyping is still indicated for patients with an abnormal ratio determined with the modified APC resistance test. The strategy described here allows us to safely lower the number of samples analysed by DGGE.