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

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.

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)

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= 30-140 increase risk of thrombosis risk factors treatment

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)

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

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

Nucleic Acid Isolation

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

LightCycler Schematic

PCR process

Detection FRET (fluorescence resonance energy transfer)

Genotyping Melting Curve Analysis

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