Automated detection of the factor V Leiden mutation using the LCx microparticle enzyme immunoassay
Harvard University, Cambridge, Massachusetts, United StatesClinical Chemistry (Impact Factor: 7.91). 01/1999; 45(1):41-6.
The factor V Leiden mutation, a G-->A transition at position 1691 in exon 10 of the gene that codes for factor V, produces an Arg506Gln substitution and is the most common genetic risk factor for venous thrombosis. We have developed a rapid, sensitive, and specific method to detect the factor V Leiden mutation in genomic DNA from whole blood by PCR amplification and microparticle enzyme immunoassay detection using the Abbott LCx instrument. We compared this automated method with the standard procedure using restriction endonuclease digestion of PCR products followed by gel electrophoresis in blinded experiments. In 130 patients (from Veterans Affairs medical centers) with deep venous thromboses, including 24 heterozygotes with the factor V Leiden mutation, there was complete agreement between the two methods. The assay was also able to distinguish heterozygotes from homozygotes. This method, which carries a low potential for cross-contamination of samples, should be a useful routine test for the factor V Leiden mutation in clinical laboratories with sufficient demand for molecular diagnostic assays using the LCx instrument.
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ABSTRACT: With the identification of common single locus point mutations as risk factors for thrombophilia, many DNA testing methodologies have been described for detecting these variations. Traditionally, functional or immunological testing methods have been used to investigate quantitative anticoagulant deficiencies. However, with the emergence of the genetic variations, factor V Leiden, prothrombin 20210 and, to a lesser extent, the methylene tetrahydrofolate reductase (MTHFR677) and factor V HR2 haplotype, traditional testing methodologies have proved to be less useful and instead DNA technology is more commonly employed in diagnostics. This review considers many of the DNA techniques that have proved to be useful in the detection of common genetic variants that predispose to thrombophilia. Techniques involving gel analysis are used to detect the presence or absence of restriction sites, electrophoretic mobility shifts, as in single strand conformation polymorphism or denaturing gradient gel electrophoresis, and product formation in allele-specific amplification. Such techniques may be sensitive, but are unwielding and often need to be validated objectively. In order to overcome some of the limitations of gel analysis, especially when dealing with larger sample numbers, many alternative detection formats, such as closed tube systems, microplates and microarrays (minisequencing, real-time polymerase chain reaction, and oligonucleotide ligation assays) have been developed. In addition, many of the emerging technologies take advantage of colourimetric or fluorescence detection (including energy transfer) that allows qualitative and quantitative interpretation of results. With the large variety of DNA technologies available, the choice of methodology will depend on several factors including cost and the need for speed, simplicity and robustness.
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ABSTRACT: The Factor V Leiden mutation is an important human polymorphism, responsible for increased risk of venous thrombosis in heterozygotes as well as homozygotes. Therefore, screening is a useful possibility, and many detection systems have been described for PCR products. We have developed a simplified and robust assay using oligonucleotide probes for normal and mutant sequences, labeled with europium and samarium, respectively, and measured by time-resolved fluorescence. Populations consisting of 233 Welsh and 148 Irish subjects were examined by both restriction fragment length polymorphism (RFLP) analysis and our assay. The allele frequency was 14/466 in the Welsh and 5/296 in the Irish population, in line with other surveys of European populations. Results were not obtained in 2/381 samples by RFLP, compared with 1/381 with our method. We conclude that our method represents an improved system capable of considerable throughput at reasonable cost.