Table 2Analytic validity of testing for the FVL mutation

Experimental TestFlap Endonuclease + FRET (Invader Assay)FRET + Melting Curve Analysis (Light Cycler)Taqman Real-Time PCR AssayElectrochemical GenosensorsTemperature Gradient Capillary ElectrophoresisMALDI-TOF Mass SpectrometryPCR-Single Strand Conformation PolymorphismSimultaneous Allele-Specific Amplification Followed by RFLPFluorogenic Probe-Based Allelic Discrimination PCR AssayMelting Analysis of Labeled Probes SystemLanthanide-Labeled ProbeRolling Circle Amplification Assay Using Open Circle Probes LigationHigh-Resolution Melting Analysis of Small AmpliconPyro-sequencingREADIT SYSTEM (Pyrophosphorolysis)ELISAReverse Allele-Specific Oligonucleotide (ASO) Hybridization AssayFirst Nucleotide Change TechnologyLinked Linear Amplification -ASO CaptureNanochip Electronic MicroarrayPhoto-Cross-Linking Oligonucleotide Hybridization AssayMGB-NFQ Probes (7700 SDS)Single-and Dual-Labeled HyBeacon Probes
Reference StandardAS-PCR or PCR-RFLPPCR or PCR-RFLPPCR-RFLPPCR-RFLPPCR-RFLPPCR-RFLPPCR-RFLPPCR-RFLPPCR-RFLPPCR-RFLP or Conventional Dual-Hybridization Probe GenotypingPCR-RFLPPCR-RFLPLight CyclerLightCycler or Direct SequencingPCR-RFLP or Not SpecifiedDirect Sequencing or PCR-RFLPPCR-RFLPPCR-RFLPPCR-RFLP & PCR-ASOPCR-RFLP, Light Cycler, Invader Monoplex AssayPCR-Based AssayFRET + Melting Curve Analysis (LightCycler)RFLP or the Roche FVL Hybridization Probe Kit
StudiesHessner, 200043 Ledford, 200044 Patnaik, 200467Schroell-Metzger, 200368 Mammo, 200669 Cooper, 200370 Nauck, 200045
Parks, 200148
Louis, 200462
Benson, 200147
Behrens, 200471
Ozkan, 200265 Ozsoz, 200372 Huang, 200273Murphy, 200361Hung, 200250 Humeny, 200155Simundic, 200374DelRio-LaFreniere, 200164Sanders, 200075El Housni, 200380 (dual-labeled probes); Vaughn, 200481 Crockett, 200182 (single-labeled fluorophore-based systems)Potter, 200149Alsmadi, 200383Liew, 200476Verri, 200577Tsongalis, 200184
Rhodes, 200159
Lopez, 200766
Carmi, 200485
Kowalski, 200058
Leyte, 200086
Pecheniuk, 200046Reyes, 200178Schrijver, 200387 Erali, 200388 Evans, 200289French, 200490Castley, 200579French, 200891
Total Population*1369 + 371 + 368132 + 2131 + 200 + 110 + 155115 + 100 + 10090 + 90 + NR30427 + 7015049120100 + 100 + 100379216104100280 + 510264 + 284256 + 99500111197 for the comparison to RFLP and 195 + 224 + 758 for the comparison to LightCycler1054151> 500
Concordance99.5% – 100%100%100%93% – 100%99.3%100%100%100%100%100%99–100%99.4%100%100%99–100%95–100%100%100%100%98–100%98.6%100%100%
Source of Zygosity Discordance**5 samples were typed WT by reference standard but heterozygous by Invader assay. With retyping, there was concordance, with all genotypes unequivocally WT. In Ledford, 2 samples were discordant. Retesting yielded results concordant with the original PCR-RFLP results. In one case, a heterozygous sample was called homozygous by the Invader assay; the discrepancy was due to a failure to add genomic DNA to the well. In the second case, a heterozygous sample was called WT.NANAIn Ozkan et al., the source and nature of the 7% discordance were not explained. Ozsoz et al. reported neither reference standard nor quantitative results. Rather, it stated that there was "good agreement" and ongoing work to achieve 100% accuracy. Huang et al. did not report sample size or reference standard.One heterozygous sample falsely identified as homozygous mutant by TGCE. One WT/WT falsely reported as WT/Leiden by TGCE.NANANANANANot reported, but at least 4 samples (1%) were discordant on the first test, and 1 (0.26%) remained discordant on repeat testing.Not reportedNANAUnclear (in Tsongalis et al., one discordant result was due to an equivocal relative light unit, while the other was indeterminate); NA59No explanation was given as to the source of the discrepancies in Lopez et al.; NA85NANANANA87, 88; In Evans et al., the reference test did not correctly identify 17 samples (details as in footnote).2 false-negatives: For one, the reference indicated homozygous, but the experimental reported heterozygous. For the other, the reference indicated heterozygous, but the experimental test indicated WT. Repeat testing confirmed the results of the reference standard for both.
False-positives: Reference indicatedWT, but the experimental test reported heterozygous initially. Repeat testing confirmed WT.
NA
Did Repeating Resolve the Discordance*?Yes (technical/operator error)NANANot reportedYes for the heterozygous; did not resolve for the WT/WT (see footnote)NANANANANAYes, but not allYes (if tests were performed in triplicate).NANAYes84; NA59YesNANANANA87, 88; No89Yes; 15 samples were discordant. All 15 samples were genotyped correctly (i.e., 100% concordance) upon retesting (reextraction of genomic DNA; resolution of the indeterminate, invalid, and discordant samples).NAn/a
*

the order of the counts corresponds to the order of the studies in the row above;

**

if <100% concordance ASO = allele-specific oligonucleotide; AS-PCR = allele-specific PCR; DNA = deoxyribonucleic acid; ELISA = enzyme-linked immunosorbent assay; FRET = fluorescence resonance energy transfer; FVL = Factor V Leiden; MALDI-TOF = matrix-assisted laser desorption/ionization – time of flight; MGB-NFQ = minor groove–binding non-fluorescent quencher; NA = not applicable; NR = not reported; PCR = polymerase chain reaction; READIT = reversed enzyme activity DNA interrogation test; RFLP = restriction fragment length polymorphism; SDS = sequence detection system; TGCE = temperature gradient capillary electrophoresis; WT = wild-type

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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