Table 2.
Summary of Platform Evaluation
| eSensor | InPlex | OLA | Signature | Tag-It | |
|---|---|---|---|---|---|
| % Concordance | 100 | 100 | 100 | 100 | 100* |
| % No calls | 0.7 | 0.7 | 0.7 | 0.7 | 0.0* |
| Signal to noise ratio | NA† | NA† | NA† | 10:1‡ | 20:1 to 100:1‡ |
| Mutations§ | Total: 23 ACMG/ACOG 23 | Total: 42 ACMG/ACOG 23 and V520F, 3876delA, 394delTT, R347H, I148T, 1078delT, 3905insT, S549N, Y122X, Y1092X S549R(T>G), 2183AA>G, S549R(A>C), D1152H, 3849 + 4A>G,E60X, Q493X, D1270N, Y1092X(C>G) | Total: 32 ACMG/ACOG 23 and V520F, 3876delA, 394delTT, R347H, I148T, 1078delT, 3905insT, S549N/R | Total: 23 ACMG/ACOG 23 | Total: 40 ACMG/ACOG 23 and V520F, 3876delA, 394delTT, R347H, I148T, 1078delT, 3905insT, S549N, Y122X, S549R(T>G), 2183AA>G, Y1092X, 2307insA, A559T, 1898 + 5G>T, M1101K, S1255X |
| Reflex tests: are poly-T reflex tests masked or run separately? | Masked¶ | Masked | Separately | Separately | Masked |
| Does the assay detect interfering benign variants (I506V, I507V, F508C) in the case of unexpected ΔF508 homozygosity? | NA∥ | NA** | Yes | Yes | Yes |
| Input DNA range (ng)†† | 10 to 600 | 25 to 350 | 1 to 15 | 10 to 1000 | 2 to 200 |
| Extraction methods used‡‡ | MP,G | MP,G | MP,G | MP,G | MP,G |
| Start to finish time (hours)§§ | 6 to 7 | 3.5 to 4 | 6 to 7 | 5 to 6 | 6.5 to 8 |
| Hands-on time (hours)§§ | 2.5 | <1 | 1.5 | 1 | 1.5 to 2.5 |
| Number of sample transfers | 3 | 2 | 2 | 2 | 4 |
| Ease of protocol¶¶ | 2 | 1 | 2 | 2 | 3 |
| Open platform∥∥ | No | No | Yes | Yes | Yes |
| Required instrumentation specific to assay | eSensor 4800 | GeNios or GeniosFL fluorometer, card bucket and clips, card sealer | ABI Prism 3100/3130 genetic analyzer | Luminex 100xMAP system | Luminex 100xMAP system |
| IVD or ASR | IVD*** | ASR | ASR | ASR | IVD |
NA, not applicable; ACMG, American College of Medical Genetics; ACOG, American College of Obstetrician and Gynecologists; MP, MagNa Pure LC DNA isolation kit 1; G, Gentra Generation capture column kit; IVD, In Vitro Diagnostic; ASR, analyte-specific reagent.
Data reported here were generated by a Tm Bioscience technician performing the assay in our laboratory. Modifications to the research use-only protocol (RUO) were made by the Tm technician. The RUO was the only available protocol at the time this study was performed. These modifications were made in an effort to minimize the previously high no-call rate and shorten the start-to-finish time and hands-on time. The modifications are as follows: 1) Exo-Sap denaturation was run 30 seconds instead of 15 minutes; 2) multiplex ASPE annealing was performed at 56°C instead of 52°C; 3) ASPE extension was run for 30 seconds instead of 1 minute; and 4) bead hybridization at 37°C was run 30 minutes instead of 1 hour.
The concepts of signal and noise do not apply to these assays.
Signal to noise ratio was calculated as follows: for Tag-It, signal is the median fluorescence intensity (MFI) generated by an allele in the sample and noise is the MFI generated by allele in the no template control; for Signature, signal is the allele ratio (mutant signal/mutant signal + wild-type signal) and noise is the standard deviation of the allele ratio.
The following platforms were able to resolve a G551D/R553X compound heterozygote: InPlex, TagIt, Signature, and eSensor. The study evaluated only the ACMG/ACOG panel of 23 mutations (2184delA was not represented). Mutations in italics are unique to that IVD/ASR.
Software for unmasking reflex test results was not available at time of evaluation; poly-T reflex test results were not evaluated.
The polymorphisms do not interfere with panel mutation genotyping in the eSensor method (information provided by the manufacturer). We did not test this aspect of the platform.
Because of the Invader chemistry, ie, the specificity with which the cleavage products are produced, the I506V and I507V polymorphisms do not interfere with genotyping (information provided by the manufacturer). We did not test this aspect of the platform. The F508C variant is assayed during the initial run with results masked.
Information provided by manufacturer.
MagNa Pure LC DNA isolation kit 1- and Gentra Generation capture column kit-extracted samples were tested on each platform. No other extraction methods were tested.
Time determined was averaged throughout several runs for 24 samples/run, excluding DNA extraction. Time calculations were based on use of a PTC-200 DNA engine thermal cycler (MJ Research/Bio-Rad). Tag-It start to finish time reflects protocol modifications indicated in *, ie, ASPE extension was run for 30 seconds instead of 1 minute; bead hybridization at 37°C was run 30 minutes instead of 1 hour.
As determined by our laboratory based on number of steps in the protocol, tolerances within those steps, and number of sample transfers.
Facilitates custom test development.
eSensor is FDA-cleared for carrier testing.