JMD 2000, Vol. 2, No. 3
Copyright © 2000 American Society for Investigative Pathology & Association for Molecular Pathology
Analytical Evaluation of Primer Engineered Multiplex Polymerase Chain Reaction–Restriction Fragment Length Polymorphism for Detection of Factor V Leiden and Prothrombin G20210A
Suzanne Huber,
Karolyn J. McMaster and
Karl V. Voelkerding
From the Department of Pathology and Laboratory Medicine, University of Wisconsin Medical School, Madison, Wisconsin
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Abstract
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Factor V Leiden and prothrombin G20210A are clinically
relevant genetic risk factors for venous thrombosis. Analysis for both
mutations is increasingly being performed on patients exhibiting
hypercoagulability. The goal of the current study was to evaluate the
performance of primer-engineered multiplex polymerase chain
reaction–restriction fragment length polymorphism (PCR-RFLP) for the
simultaneous detection of factor V Leiden and prothrombin G20210A.
Primer-engineered multiplex PCR-RFLP methods for the detection of
factor V Leiden and prothrombin G20210A from the medical literature
were reviewed. A modified method was optimized in which both mutations
generate HindIII RFLPs and the prothrombin amplicon
contains an invariant HindIII recognition site to assess
the completeness of endonuclease digestion. Digested amplification
products were analyzed by agarose gel electrophoresis in a single gel
lane and visualized by ethidium bromide. Primer-engineered multiplex
PCR-RFLP was used to analyze 205 human genomic DNA samples whose factor
V Leiden genotypes had been previously determined by
MnlI PCR-RFLP. Complete concordance for factor V Leiden
genotypes was observed between the two methods in the 205-sample cohort
comprising 139 wild-type, 62 heterozygous mutant,
and four homozygous mutant individuals. For prothrombin
G20210A, primer-engineered multiplex PCR-RFLP identified 196
wild-type and nine heterozygous mutant individuals in the 205-sample
cohort. To independently verify prothrombin genotypes, the nine
heterozygous mutants and an additional 11 wild-type patient
samples (representing 10% of patient samples) were subjected to DNA
sequencing. Complete concordance was observed between DNA sequencing
and primer-engineered multiplex PCR-RFLP results. In further
validation, 123 of the DNA samples consisting of four
heterozygous mutant and 119 wild type individuals were genotyped with
the Invader Assay for Factor II (prothrombin G20210A). Results showed
100% concordance between the Invader AssayTM and primer-engineered
multiplex PCR-RFLP. A primer-engineered multiplex PCR-RFLP based on
single restriction endonuclease digestion has been evaluated and shown
to simultaneously and accurately detect factor V Leiden and prothrombin
G20210A mutations. The method is robust and readily adaptable to the
clinical molecular diagnostic laboratory.
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Introduction
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Genetic risk factor assessment has become an integral component of the
diagnostic evaluation of patients presenting with signs and symptoms of
venous thrombosis. The most common known genetic risk factor associated
with venous thrombosis is resistance to activated protein C, which, in
the majority of cases, is due to a single G-to-A nucleotide transition
in exon 10 of the factor V gene that results in the replacement of
arginine, at amino acid position 506, with glutamine (factor V
Arg506Gln or factor V Leiden).1, 2
The factor V Leiden mutation is found in Caucasians, and its prevalence
varies from 2% to 13%, depending on geographic locale and ethnic
composition. Heterozygotes for factor V Leiden have an approximate
eightfold increased relative risk for the development of venous
thrombosis, and homozygotes are estimated to have an approximately
90-fold increased relative risk.3
The identification of factor V Leiden in 1994 was followed in 1996 by
the elucidation of a single G-to-A transition at position 20210 of the
prothrombin gene. The G20210A mutation occurs in the 3' untranslated
region of the prothrombin gene just before the site of polyadenylation.
Heterozygous carriers of prothrombin G20210A exhibit higher mean plasma
prothrombin concentrations, and increased prothrombin levels are
hypothesized to be a risk factor for venous thrombosis. The prothrombin
G20210A mutation is present in approximately 2% of healthy Caucasians
and 6% of patients with a first episode of venous thrombosis. The
prothrombin G20210A mutation increases the relative risk of venous
thrombosis by approximately threefold.4
Molecular diagnostic testing for factor V Leiden is widespread, and
laboratories use a variety of technical approaches. An increasing
number of laboratories are additionally analyzing for prothrombin
G20210A. Several authors have reported on multiplex polymerase chain
reaction–restriction fragment length polymorphism (PCR-RFLP)
approaches for the combined detection of both factor V Leiden and
prothrombin G20210A.5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16
In the current study, multiplex
PCR-RFLP approaches in the medical literature were reviewed, and a
modification of reported approaches was established based on
primer-engineered multiplex PCR-RFLP. We evaluated the multiplex method
for its ability to detect factor V Leiden and prothrombin G20210A in a
205-patient base sample. We determined that the method performed
robustly and accurately.
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Materials and Methods
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Genomic DNA was isolated using the Epicentre Master Pure Genomic
DNA purification kit (Madison, WI). Use of residual human genomic DNAs
in this study was approved by the University of Wisconsin Hospital and
Clinics Institutional Review Board. A 241-bp product from exon 10 of
factor V (GenBank accession L32764) and a 506-bp product from the 3'
untranslated region of the prothrombin gene (GenBank accession M17262)
were coamplified using PCR. Mismatched antisense primers were used to
amplify both products. Factor V primers were as follows: sense, 5'-TCA
GGC AGG AAC AAC ACC AT-3'; antisense, 5'-GGT TAC TTC AAG GAC AAA ATA
CCT GTA AAG CT-3'.17
In the
antisense primer, three mismatched nucleotides (underlined) were
substituted so that amplification of a mutant allele resulted in the
generation of a new HindIII restriction endonuclease site.
Prothrombin primers were as follows: sense, 5'-GCA CAG ACG GCT GTT CTC
TT-3'; antisense, 5'-ATA GCA CTG GGA GCA TTG AAG
C-3'.8
Similarly, a single mismatched nucleotide
(underlined) was replaced in the antisense primer, so that
amplification of a mutant allele would result in the creation of a
HindIII site. Genomic DNA (250 ng) was added to a PCR mix
containing 1x Gene Amp Buffer (10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L
KCl, 1.5 mmol/L MgCl2, 0.001% w/v gelatin; Roche
Molecular Systems, Branchburg, NJ), 0.2 mmol/L (each) dNTP mix
(Promega, Madison, WI), 1.2 µmol/L factor V primers, 0.1 µmol/L
prothrombin primers, and 2.5 U AmpliTAQ Gold (Roche Molecular Systems)
in a final volume of 50 µl. DNA was amplified in a thermal cycling
reaction consisting of a 10-minute enzyme activation at 95°C followed
by 40 cycles of 94°C for 1 minute, 55°C for 1 minute, and 72°C
for 1 minute with a final 5-minute extension at 72°C. A prothrombin
G20210A heterozygote and a factor V Leiden heterozygote were used as
controls in each run. Water (reagent) controls were included at the
beginning and end of each run. Twenty-six microliters of amplified
products was then digested with 1 µl HindIII (60,000 U/ml;
Promega) with 3 µl of 10x enzyme buffer at 37°C for either 2 hours
or overnight. Digested and undigested products were separated in a 20
cm x 27 cm x 7 mm, 3% agarose gel containing 0.5 µg/ml
ethidium bromide for 840 V-hours. Undigested products resulted in two
products of 506 and 241 bps, representing prothrombin and factor V
amplicons, respectively. HindIII digestion of factor V and
prothrombin wild-type amplicons yielded fragments of 241 bp (factor V
amplicon) and 407 bp and 99 bp (prothrombin amplicon); the 99-bp
fragment was a result of an invariant HindIII site.
Digestion of the factor V Leiden heterozygote resulted in fragments of
241 bp, 209 bp, and 32 bp, and the prothrombin G20210A heterozygote
yielded fragments of 407 bp, 384 bp, 99 bp, and 23 bp. Factor V Leiden
and prothrombin 20210A homozygotes digested with HindIII
yielded fragments of 384 bp, 99 bp, and 23 bp (prothrombin amplicon)
and 209 bp and 32 bp (factor V amplicon). For DNA sequencing of
prothrombin alleles, a 392-bp product from exon 14 and the 3' UTR of
the prothrombin gene that encompasses the G20210A mutation was
amplified using the primers of Poort et al (5'-TCT AGA AAC AGT TGC CTG
GC-3' and 5'-TCC AGT AGT ATT ACT GGC TC-3').18
This
fragment was sequenced using the ABI Prism Dye terminator cycle
sequencing ready kit (PE Applied Biosystems, Foster City, CA) and the
Applied Biosystems 373 DNA Sequencer. Sequencing results were surveyed
for heterozygosity with MT Navigator software (PE Applied Biosystems).
The Invader AssayTM for Factor II (prothrombin) was performed according
to the manufacturer’s instructions (Third Wave Technologies, Madison,
WI).19, 20
A total of 5 µl of Invader Reaction Mix (4
µl 16% polyethylene glycol/50 mmol/L 3-(N-morpholino)propanesulfonic
acid and 1 µl 0.5 mmol/L Invader oligo of sequence
5'-TATGGTTCCCAATAAAAGTGACTCTCAGCT-3') was pipetted into each well of a
96-well microplate. Ten microliters of DNA sample (200 ng DNA) was
added and mixed. The following synthetic oligonucleotide target
controls were tested for both wild-type and mutant probes: no DNA
target (for background determination), wild type, homozygous mutant,
and heterozygous mutant. Fifteen microliters of Chill-outTM 14 (MJ
Research, Watertown, MA) was overlaid on each well. The plate was
heated in a PTC-100 thermal cycler (MJ Research) at 95°C for 5
minutes and cooled to 63°C. Then 5 µl Cleavase/Probe Reaction Mix
(1 µl of 10 mmol/L wild-type probe or mutant probe, 2 µl 75 mmol/L
MgCl2, 1 µl 10 mmol/L 1-Piece Fret Oligo, and 1
µl 200 ng/ml cleavase VIII) was added, mixed, and incubated at 63°C
for 4 hours. Probe sequences are were as follows: factor II wild type,
5'-AACGAGGCGCACGAGCCTCAATGCTCCC-3'; factor II mutant,
5'-AACGAGGCGCACAAGCCTCAATGCTCCC-3'; fluorescence resonance energy
transfer (FRET) probe,
5'-F-CCTC-Q-GTCTCGGTTTTCCGAGACGAGGGTGCGCCTCGTTT-3', where F is a
fluorescein dye and Q is a quenching dye, Cy3. Reactions were stopped
by adding 100 µl of 10 mmol/L EDTA. One hundred microliters of each
reaction was transferred to a FluoroNunc microtiter plate, and the
fluorescent emission was measured in a CytoFluor Series 4000
Fluorescence MultiWell Plate Reader (PerSeptive Biosystems, Framingham,
MA) or a Dynex Fluorite 1000 Fluorescence MultiWell Plate Reader (Dynex
Technologies, Chantilly, VA). Settings for the CytoFLuor Series 4000
were excitation 485/20 nm (wavelength/bandwidth), emission 530/25 nm
(wavelength/bandwidth), and gain 50, which yielded no target counts
between 100 and 200. Settings for the Dynex Fluorite 1000 were
excitation 485 nm, emission 530 nm, and gain 2.6–4.0, which yielded no
target counts between 100 and 200.
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Results
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A modified primer-engineered multiplex PCR method was optimized to
result in the coamplification of 241-bp factor V and 506-bp prothrombin
amplicons that contain the Leiden and G20210A mutation sites,
respectively. HindIII-digested amplification products were
separated by electrophoresis on 3% agarose gels and visualized by
ethidium bromide. A representative multiplex analysis is shown in
Figure 1
. To evaluate the multiplex method, 205 residual human genomic DNA
samples that had previously been genotyped in our laboratory by
MnlI PCR-RFLP were analyzed.21
Complete
concordance for factor V Leiden genotypes was observed between the two
methods in the sample cohort comprising 139 wild-type, 62 heterozygous
mutant, and four homozygous mutant individuals. The multiplex
analytical results indicated that of the 205 samples, nine were
heterozygous mutant for prothrombin G20210A, and the remaining 196
samples were wild type. To independently verify prothrombin results,
DNA sequencing was performed on the nine heterozygous mutants and an
additional 11 wild-type samples. These 20 samples, representing 10% of
the sample base, showed complete concordance between DNA sequencing and
multiplex PCR results. In further validation, 123 of the DNA samples,
consisting of four heterozygous mutant and 119 wild type, were
genotyped using the Invader Assay for Factor II (prothrombin
G20210A).19, 20
Results showed 100% concordance between
the Invader AssayTM and primer-engineered multiplex PCR-RFLP.
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Figure 1. Photograph of ethidium bromide-stained 3% agarose gel demonstrating
primer-engineered PCR analysis for prothrombin G20210A and factor V
Leiden. M denotes the molecular weight marker; W denotes water control.
Lane 1: Undigested wild-type 506-bp
(prothrombin) and 241-bp
(factor V) amplicons.
Lane 2: A prothrombin G20210/factor V wild type with
digested products of 407, 241, and 99 bp. Lane 3: A
prothrombin G20210 wild-type/factor V Leiden heterozygote with
digestion products of 407, 241, 209, and 99 bp
(32-bp product not
shown). Lane 4: A prothrombin G20210
wild-type/factor V Leiden homozygote with digestion products of 407,
209, and 99 bp (32-bp product not
shown). Lane 5: A prothrombin G20210A
heterozygote/factor V Leiden heterozygote with digestion products of
407, 384, 241, 209, and 99 bp (32- and 23-bp
products are not shown).
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Discussion
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The aim of the current study was to establish in our molecular
diagnostic laboratory a method for the simultaneous detection of factor
V Leiden and prothrombin G20210A. In reviewing the literature, we
elected to combine features of several methods to achieve a multiplex
PCR approach based on primer-engineered RFLP detection of factor V
Leiden and prothrombin G20210A.8, 17
Central to the
approach presented is the use of a single restriction endonuclease
(HindIII) and incorporation of an invariant
HindIII recognition site in the prothrombin amplicon to
assess the completeness of endonuclease digestion. Presumably because
of the three-base mismatch in the factor V antisense primer, we
observed significantly less amplification of the factor V amplicon
compared to the prothrombin amplicon when equivalent primer
concentrations were used. We therefore titrated primer concentrations
to arrive at a 12:1 ratio of factor V to prothrombin primers, which
resulted in approximately equal amplification of the two amplicons.
For evaluation of the multiplex PCR method, 205 archived genomic DNA
samples were analyzed. All samples had previously been genotyped for
factor V Leiden in our laboratory, using a PCR-RFLP method based on
MnlI recognition.21
We observed complete
concordance between methods. Analysis for prothrombin G20210A
identified nine heterozygotes in the 205-patient sample cohort; the
remainder exhibited wild-type genotypes. We elected to sequence the
nine heterozygotes and an additional 11 wild-type samples and observed
complete concordance. Although we did not sequence all 205 samples for
prothrombin G20210A, our survey of 10% of the samples, including those
identified as heterozygotes, provides an independent
measure of assay accuracy. In further validation, 123 of
the DNA samples consisting of four heterozygous mutant and 119 wild
type were genotyped with the Invader AssayTM for Factor II
(prothrombin G20210A). Results showed 100% concordance between the
Invader AssayTM and primer-engineered multiplex PCR-RFLP.
The current multiplex PCR relies on the generation of novel
HindIII recognition sites for demonstration of the Leiden
and G20210A mutations. One can envision a scenario in which a second
mutation or polymorphism at the site of primer hybridization prevents
the generation of the HindIII site, a situation that may
yield a false negative result. An additional possibility is that a
polymorphism at the site of primer hybridization could result in
nonamplification of an allele. This latter possibility would likely be
most relevant to the factor V Leiden portion of the assay because of
the inherent instability of the 3-bp mismatched antisense primer. Two
apparently rare polymorphisms flanking the G1691A Leiden mutation have
been reported (G1689A and A1692C).22, 23, 24
The former polymorphism would not affect the current multiplex PCR. The
A1692C polymorphism would potentially affect the multiplex PCR. First,
A1692C would cause a noncontiguous 4-bp mismatch (comprising of the
3-bp mismatch of the factor V antisense primer and that of the A1692C
polymorphism). This degree of mismatch might result in loss or
reduction of allele amplification. Second, if allele amplification were
to proceed in the presence of A1692C, the polymorphism would likely
preclude generation of a HindIII site in the presence of
factor V Leiden. These two effects of the A1692C polymorphism could
generate either false positive or false negative results, depending on
the Leiden genotype of the individual.
The frequency of A1692C has been estimated at less than
0.025%.23, 24
Although this represents a relatively rare
polymorphism, it should be considered to be a limitation of the
multiplex PCR. Interestingly, A1692C generates a false positive result
for factor V Leiden in the widely used MnlI-based PCR-RFLP.
The multiplex method will allow us to provide genotypic information on
the prothrombin locus at no additional cost or labor over that required
for factor V Leiden genotyping by Mnl I PCR-RFLP. A modest
cost savings is realized because of the lower cost of
HindIII compared to Mnl1. The multiplex method
generates genotypic information for two distinct loci. This raises
complex issues pertaining to the generation of genetic information and
specifically how to operationalize multiplex genetic assays when
diagnostic information is sought for only one of the loci interrogated
by the assay. In the setting of hypercoagulability assessment, current
clinical practice is to evaluate multiple risk factors. In this regard,
testing for both Leiden and prothrombin mutations is increasing. As the
effort and cost of validation as described in the current study are
considerable, we determined that referring physicians were interested
in knowing the status of both Leiden and prothrombin mutations in their
patients being evaluated for hypercoagulability. However, this approach
may not be desired by all institutions and would have to be
individually addressed by each laboratory.
In summary, the primer-engineered multiplex PCR-RFLP evaluated in this
study performed robustly and accurately and is readily adaptable to the
clinical molecular diagnostic laboratory setting.
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Footnotes
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Address reprint requests to Dr. Karl V. Voelkerding, GeneInsight, 609 Sprague Street, Madison, WI 53711. E-mail: voelker{at}geneinsight.org
Supported by a University of Wisconsin Applied Research Grant (to K. V. V.).
Dr. Huber’s present address is Waisman Center, University of
Wisconsin, Madison, WI 53792.
Accepted for publication May 30, 2000.
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References
|
|---|
-
Bertina RM, Koeleman BPC, Koster T, Rosendaal FR, Dirven RJ, de Ronde H, van der Velden PA, Reitsma PH: Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature 1994, 369:64-67[Medline]
-
Dahlbäck B: Procoagulation and anticoagulant properties of coagulation factor V: factor V Leiden (APC resistance) causes hypercoagulability by dual mechanisms. J Clin Lab Med 1999, 133:415-422[Medline]
-
Bertina RM: Factor V Leiden and other coagulation factor mutations affecting thrombotic risk. Clin Chem 1997, 43:1678-1683[Abstract/Free Full Text]
-
Poort SR, Rosendaal FR, Reitsma PH, Bertina RM: A common genetic variant in the 3'-untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increase in venous thrombosis. Blood 1996, 88:3698-3703[Abstract/Free Full Text]
-
Giansily M, Biron C, Jeanjean P, Masmejean C, Schved JF, Aguilar PM: An even easier method for one-step detection of both FV Leiden and FII G20210A transition. Blood 1998, 92:3478-3479[Free Full Text]
-
Gomez E, van der Poel SC, Jansen JH, van der Reijden BA, Lowenberg B: Rapid simultaneous screening of factor V Leiden and G20210A prothrombin variant by multiplex polymerase chain reaction on whole blood. Blood 1998, 91:2208-2209[Free Full Text]
-
Wisotzkey JD, Bell T, Monk JS: Simultaneous polymerase chain reaction restriction fragment length polymorphism identification of the factor V Leiden allele and the prothrombin 20210A mutation. Diagn Mol Pathol 1998, 7:180-183[Medline]
-
Danneberg J, Abbes AP, Bruggeman BJ, Engel H, Gerrits J, Martens A: Reliable genotyping of the G-20210-A mutation of coagulation factor II (prothrombin). Clin Chem 1998, 44:349-351[Free Full Text]
-
Ripoll L, Paulin D, Thomas S, Drouet LO: Multiplex PCR-mediated site-directed mutagenesis for one-step determination of factor V Leiden and G20210A transition of the prothrombin gene. Thromb Haemost 1997, 78:960-961[Medline]
-
Linfert DR, Rezuke WN, Tsongalis GJ: Rapid multiplex analysis for the factor V: Leiden and prothrombin G20210A mutations associated with hereditary thrombophilia. Conn Med 1998, 62:519-525[Medline]
-
Xu X, Bauer KA, Griffin JH: Two multiplex PCR-based DNA assays for the thrombosis risk factors prothrombin G20210A and coagulation factor V G1691A polymorphisms Thromb Res 1999, 93:265-269[Medline]
-
Mitterer M, Lanthaler AJ, Mair W, Giacomuzzi K, Coser P: Simultaneous detection of FV Q506 and prothrombin 20210A variation by allele-specific PCR. Haematologica 1999, 84:204-207[Abstract/Free Full Text]
-
Keeney S, Salden A, Hay C, Cumming A: A whole blood, multiplex PCR detection method for factor V: Leiden and the prothrombin G20210A variant. Thromb Haemost 1999, 81:464-465[Medline]
-
Raoul M, Mathonnet F, Peltier JY, Collet C, Boucly C, Van Amerongen G, Mathieu B, Jaouen E, de Mazancourt P: An improved method for the detection of the G20210A transition in the prothrombin gene. Thromb Res 1997, 88:441-443[Medline]
-
Poort SR, Bertina RM, Vos HL: Rapid detection of the prothrombin 20210 A variation by allele specific PCR. Thromb Haemost 1997, 78:1157-1158[Medline]
-
Lastrucci RMD, Dawson DA, Bowden JH, Munster M: Development of a simple multiplex polymerase chain reaction for the simultaneous detection of factor V Leiden and prothrombin 20210A mutations. Mol Diagn 1999, 4:247-250[Medline]
-
Gandrille S, Alhenc-Gelas M, Aiach M: A rapid screening method for The factor V Arg506
Gln mutation. Blood Coagul Fibrinolysis 1995, 6:245-248[Medline]
-
Poort SR, Michiels JJ, Reitsma PH, Bertina RM: Homozygosity for a novel missense mutation in the prothrombin gene causing a severe bleeding disorder. Thromb Haemost 1994, 72:819-824[Medline]
-
Lyamichev V, Mast AL, Hall JG, Prudent JR, Kaiser MW, Takova T, Kwiatkowski RW, Sander TJ, de Arruda M, Arco DA, Neri BP, Brow MA: Polymorphism identification and detection of genomic DNA by invasive cleavage of oligonucleotide probes. Nature Biotechnol 1999, 17:292-296[Medline]
-
Kwiatkowski RW, Lyamichev V, de Arruda M, Neri BP: Clinical, genetic and pharmacogenetic applications of the InvaderTM Assay. Mol Diagn 1999, 4:353-364[Medline]
-
Voelkerding KV, Wu L, Williams EC, Hoffman SM, Sabatini LM, Borcherding WR, Huber S: Factor V R506Q gene mutation analysis by PCR-RFLP. Am J Clin Pathol 1996, 106:100-106[Medline]
-
Leibman HA, Sutherland D, Bacon R, McGehee W: Evaluation of a tissue factor dependent factor V assay to detect factor V Leiden: demonstration of high sensitivity and specificity for a generally applicable assay for activated protein C resistance. Br J Haematol 1996, 95:550-553[Medline]
-
Bowen DJ, Standen GR: Genetic detection of factor V Leiden: the question of specificity. Br J Haematol 1997, 97:691-692[Medline]
-
Lyondagger E, Millsondagger A, Phan T, Wittwer CT: Detection and identification of base alterations within the region of factor V: Leiden by fluorescent melting curves. Mol Diagn 1998, 3:203-209[Medline]
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Abstract
Introduction
