What blood test is used to detect Factor V Leiden?

Factor V Leiden Blood Testing

Factor V Leiden is detected through direct DNA-based molecular genetic testing for the R506Q mutation in the factor V gene, which is the definitive diagnostic method recommended by the American College of Medical Genetics. 1

Primary Testing Method: DNA-Based Molecular Testing

The gold standard is direct molecular genetic testing that identifies the specific G1691A (R506Q) mutation in the factor V gene. 1 This can be performed using several validated technical approaches:

• PCR-based methods with restriction endonuclease digestion (AMP-FLPs) - highly accurate and widely used 1
• Allele-specific PCR - allows direct amplification of mutant or wild-type alleles 1
• Allele-specific oligonucleotide probe hybridization - uses probes that bind specifically to mutant or normal sequences 1
• Real-time fluorescent PCR (TaqMan assays) - provides rapid results with high sensitivity and specificity 2

The DNA test has extremely low false-negative and false-positive rates when performed properly using standard techniques. 1

Sample Requirements

• Whole blood at room temperature is required for DNA extraction and testing 1, 3
• This is simpler than functional assays which require citrated frozen plasma prepared by centrifugation 1

Alternative: Functional APC Resistance Testing

A functional activated protein C (APC) resistance assay can be used as an initial screening test, but has important limitations: 1

• Modified APC resistance assay (with dilution in factor V-deficient plasma) has high sensitivity and specificity for Factor V Leiden 1
• Traditional aPTT-based APC resistance test has high sensitivity but suboptimal specificity and is subject to interference from pregnancy, oral contraceptives, lupus anticoagulant, and acute phase reactants 1
• Cannot be used in patients on heparin or warfarin - these patients must proceed directly to DNA testing 1, 3
• Cannot reliably distinguish heterozygotes from homozygotes due to overlapping values, which is clinically critical since homozygotes have 10-fold higher thrombotic risk than heterozygotes 1

Clinical Algorithm for Test Selection

For patients on anticoagulation (heparin, warfarin, or direct oral anticoagulants like apixaban): Proceed directly to DNA-based molecular testing 1, 3

For patients with known lupus anticoagulant: Use DNA testing rather than functional assays 1

For testing relatives of known Factor V Leiden carriers: DNA testing is recommended 1

For initial screening in appropriate clinical scenarios: Either DNA testing or modified functional APC resistance assay is acceptable, but any positive functional test must be confirmed with DNA testing to distinguish heterozygotes from homozygotes 1

Important Caveats

• Factor V Leiden is the only molecular analysis of the factor V gene indicated in routine thrombotic risk workup - testing for other factor V variants like R2 (H1299R) is not routinely recommended 1
• DNA testing correctly identifies heterozygotes versus homozygotes, which is essential for risk stratification since homozygotes have approximately 80-fold increased thrombosis risk compared to 7-fold for heterozygotes 1
• Laboratory proficiency testing shows 98.9-99.6% accuracy rates for Factor V Leiden DNA testing 3
• Results typically take several days as DNA extraction and genetic analysis cannot be significantly expedited 3

Quality Assurance Standards

All Factor V Leiden mutation testing should adhere to current ACMG/CAP quality assurance guidelines for molecular genetic testing, with proper laboratory validation of the chosen methodology. 1