1. Factor V Leiden Detection and Genotyping
August 6, 2008
Marylin Bicak

2. Objective 1. To provide an overview of the Factor V Leiden assay
2. To explain the pathophysiology and epidemiology of the Factor V Leiden mutation.
3. To evaluate the efficacy of the assay.

3. Pathophysiology inherited condition autosomal dominant
chromosome 1, gene F5
Single point mutation, G1691A (arginine to glutamine at 506th amino acid
mutation prevents inactivation of factor V in clotting process (resistant to inactivation by Protein C)
overproduction of thrombin= excess fibrin formation
excess clotting (DVT and PE)

4. Epidemiology Most common inherited coagulation disorder in U.S.
5% Caucasians
2% Hispanics
1.2% African Americans
<0.5% Asian Americans
Heterozygous= 3-8 fold increase risk of thrombosis
Homozygous= increase risk of thrombosis
risk factors
treatment

5. Factor V Leiden Assay
1. Extraction- MagNA Pure Compact Nucleic Acid Isolation Kit and instrument (Roche Diagnostics)
2. Amplification/Detection/Genotyping- Factor V Leiden Kit and LightCycler 2.0 instrument (Roche Diagnostics)

6. Extraction
Specimen- EDTA whole blood (200 ul whole blood= 100 ul purified product)
MagNA Pure Kit- pre-sealed reagent cartridge; disposable pipette tip tray assembly; sample tube; elution tube
MagNA Pure Compact Instrument-automated method based on magnetic glass particle technology

7. Principle of Magnetic Glass Particle Technology
Four step process: 1-lysis; 2-bind to magnetic particles; 3-wash; 4-elution 

8. Nucleic Acid Isolation

9. Amplification-real-time PCR
target- 222 bp fragment of Factor V gene
Factor V Leiden kit- master mix reagents (primers and probes)
LightCycler 2.0 instrument- rapid- 45 cycles (30 min)
Cycle temperatures- denaturation 95C; annealing 60C; elongation 72C
thermal cycler- heat/ambient air cycle
reaction vessel- 20 ul glass capillary

10. LightCycler Schematic

11. PCR process

12. Detection
FRET (fluorescence resonance energy transfer)

13. Genotyping
Melting Curve Analysis

14. Summary
Limitations- technical process/ physical space/ instrumentation
Erroneous results- a. false positive (3 rare mutations span same mutation probe) and b. patient sample with elevated WBC
Overall, innovative system and important diagnostic tool for clinical lab