Laboratory Testing for Factor V Leiden Disorder
Order direct DNA-based genotyping for the Factor V Leiden (G1691A) mutation as the primary test, and simultaneously test for prothrombin G20210A mutation, as these are the two most common inherited thrombophilias and can be multiplexed together. 1
Primary Testing Approach
Initial Test Selection
Either direct DNA-based genotyping or a factor V Leiden-specific functional assay (activated protein C resistance) is recommended initially for diagnosing Factor V Leiden. 1
DNA testing is preferred in your patient's scenario because it definitively distinguishes heterozygotes from homozygotes in a single test, which is critical for risk stratification and management decisions. 1
The functional assay (activated protein C resistance) can be used as a screening test, but any positive result requires confirmatory DNA testing to determine zygosity status. 1
Concurrent Testing Strategy
Test for prothrombin G20210A mutation simultaneously with Factor V Leiden testing, as this is the most common co-existing thrombophilia with overlapping phenotype, present in 1-2% of the general population. 1
The DNA test for prothrombin mutation is as simple as Factor V Leiden testing and can be multiplexed with it in the same assay. 1
Patients heterozygous for both Factor V Leiden and prothrombin 20210A mutation have synergistic risk that exceeds either mutation alone and require more aggressive management. 1
Comprehensive Thrombophilia Panel
Given the strong clinical indication (family history of blood clots or recurrent thrombotic events), consider a complete thrombophilia evaluation including: 1, 2
- Factor V Leiden (G1691A) mutation - DNA testing 1, 2
- Prothrombin G20210A mutation - DNA testing 1, 2
- Antithrombin III level/activity 1, 2
- Protein C level/activity 1, 2
- Protein S level/activity (free and total) 1, 2
- Plasma homocysteine levels 1
Critical Testing Considerations
Timing of Testing
If the patient is currently on anticoagulation, proceed directly to DNA molecular testing rather than functional assays, as anticoagulation interferes with functional coagulation assays but does not affect DNA testing results. 1
Patients on heparin therapy or with known lupus anticoagulant should proceed directly to molecular testing. 1
Ideally, functional coagulation assays (Protein S, Protein C, antithrombin III) should be performed when not on anticoagulation to avoid interference, though DNA testing is unaffected by anticoagulation status. 1
Confirmatory Testing Algorithm
If using the functional assay (activated protein C resistance) as initial screening and it returns positive, confirmatory DNA testing is mandatory to distinguish heterozygotes from homozygotes, as this distinction fundamentally changes management. 1
Homozygotes should be considered for lifetime antithrombotic prophylaxis after a thrombotic event, while heterozygotes typically receive standard duration anticoagulation. 1, 3
Common Pitfalls to Avoid
Sample misidentification and faulty allele-specific amplification methods can lead to false-negative results, so if clinical suspicion remains high despite negative testing, consider repeat testing at a different laboratory. 4
In patients with prior allogeneic bone marrow transplantation, genetic testing may show disappearance of Factor V Leiden due to donor chimerism, leading to false-negative results. 5
Do not rely solely on family history to predict inherited thrombophilia, as negative family history does not exclude it due to low penetrance and possible de novo mutations. 2
Patient Counseling Before Testing
- Inform patients that thrombophilia testing is genetic, has implications for family members, and may have confidentiality and insurance implications before ordering tests. 1