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Factor V Leiden Detection and Genotyping
August 6, 2008 Marylin Bicak
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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.
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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)
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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
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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)
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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
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Principle of Magnetic Glass Particle Technology
Four step process: 1-lysis; 2-bind to magnetic particles; 3-wash; 4-elution
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Nucleic Acid Isolation
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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
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LightCycler Schematic
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PCR process
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Detection FRET (fluorescence resonance energy transfer)
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Genotyping Melting Curve Analysis
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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
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