Transcript Factor V Leiden Detection and Genotyping

Factor V Leiden
Detection and
Genotyping
August 6, 2008
Marylin Bicak
Objective
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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
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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
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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
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1. Extraction- MagNA Pure Compact Nucleic
Acid Isolation Kit and instrument (Roche
Diagnostics)
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2. Amplification/Detection/Genotyping- Factor
V Leiden Kit and LightCycler 2.0 instrument
(Roche Diagnostics)
Extraction
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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
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Four step process: 1-lysis; 2-bind to magnetic
particles; 3-wash; 4-elution
Nucleic Acid Isolation
Amplification-real-time PCR
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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
Detection
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FRET (fluorescence resonance energy
transfer)
Genotyping
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Melting Curve Analysis
Summary
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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