Table 7Details of studies on the analytic validity of simultaneous detection (multiplex) of FVL and prothrombin G20210A mutations

Study(s), Year(s)Description
Hobson-Peters, 200593Used FRET with a dual-probe quenching system that is similar to the Light Cycler assay.
Endler, 20015670 known patients with recurrent venous thrombosis and 83 selected healthy controls from the Austrian Study of Recurrent VTE for whom previous determination of the genotypes for the FV:R506Q (G1691A), the FII:G20210A, and MTHFR:A223V (C677T) variants had been established by PCR followed by RFLP
Baris, 200495No reference method was reported. For concordance, known patients with recurrent venous thrombosis with "previously determined" genotypes for FVL and prothrombin G20210A were used. FVL was amplified significantly less than prothrombinG20210A, with an initial primer concentration of 2:1. The effects of various factors, including magnesium cation concentration, primer concentration, and PCR cycling conditions, on PCR specificity and efficiency were determined and optimized in this study.
Huber, 200096Concordance was only tested on FVL. For prothrombin, the multiplex PCR-RFLP identified 9 heterozygous and 196 wild-type samples. To independently verify prothrombin results, DNA sequencing confirmed 9 heterozygous mutant and 11 wild-type samples (10% of the sample). The study also genotyped 123 samples with multiplex PCR-RFLP and the invader assay for Factor II, with 100% concordance.
Lucotte, 200397Duplex PCR-RFLP for simultaneous detection of FVL and prothrombin G20210A was used.
Koksal, 200757Significantly less amplification was noted for FV relative to prothrombin; the FV primer was therefore 2x concentrated relative to prothrombin.

DNA = deoxyribonucleic acid; FII = Factor II; FV = Factor V; FRET = fluorescence resonance energy transfer; FVL = Factor V Leiden; MTHFR = methylenetetrahydrofolate reductase; PCR = polymerase chain reaction; RFLP = restriction fragment length polymorphism; VTE = venous thromboembolism

From: 3, Results

Cover of Outcomes of Genetic Testing in Adults with a History of Venous Thromboembolism
Outcomes of Genetic Testing in Adults with a History of Venous Thromboembolism.
Evidence Reports/Technology Assessments, No. 180.
Segal JB, Brotman DJ, Emadi A, et al.

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