Highlighting problematic probe bindings
Mutation reactions can be prone to inefficient and non-spe- cific probe binding, leading to late crossing points even in the absence of a mutation. This problem can be seen in the data gathered in this study, showing false-positive results for mutations G719X and T790M in the negative controls.
20.0 22.0 24.0 26.0 28.0 30.0 32.0 34.0 36.0 38.0 40.0
20.0 22.0 24.0 26.0 28.0 30.0 32.0 34.0 36.0 38.0 40.0
T790M Exon L858R L861Q G719X
790M Exon 19del
Figure L858R 861Q 719X
Comparison of an assay’s performance between mutations The results showed a 95% confidence level in HDx Refer- ence Standards (from 17 assays) with a mean Crossing Point: +/- 0.26 (L858R) to +/- 0.56 (G719X). The adoption of the 2.5% EGFR HDx Reference Standard allows Trillium Health Partners to evaluate the workflow to a higher degree of sensitivity in addition to the in-kit positive control.
2.5%2.5% 1%
1% Ne
Neg Ctl tl
Figure 2 – Assay evaluat on specific crossing points cut-offs for EGFR L861Q
tumor content
Assay evaluation specific crossing pointsi for EGFR L861Q
Figure 3- Comparison of crossing points for EGFR mutations
Identification of mutations that are a true positive but have low percentage tumor content
true positive but have low percentage
crossing point will be high and possibly below the 2.5% established cut-off. Although the samples’ crossing points are later than that of the negative control, qualitative assessment must be used to evaluate each individual case. This can be seen in the G719X reaction example.
When the mutation is in a low percentage of cells in the sample, the crossing point will be high and possibly below the 2.5% established cut-off. the samples’ crossing points are later than that of the negative qualitative assessment must be used to evaluate each individual This can be seen in the G719X reaction example.
the mutation is in low percentage of cells in the sample, the 2.5% C 2.5% Control (31.85) Negativ Kit Control (28.24)
Sample Negative Control
Sample A (34.70)
Figure 4 – A suggested true positive by qualitative assessment at 1-2%
Figure assessment at 1-2%
Conclusions • Crossing point values alone do not tell the whole story • A high crossing point above the established cut-offs determined during assay evaluation can indicate true positives • HDx Reference Standards provide support for assay evaluation to establish key performance characteristics •
Low allelic frequency Reference Standards supply a qualitative reference for true amplification About Horizon Diagnostics
Horizon Diagnostics is proud to support laboratories with the provision of reference materials for research use. Applications include the evaluation of equipment and consumables offline from patient testing. This report has been provided for information only. To find out more please visit
www.horizondx.com. BioTechniques 58:268-269 (May 2015) doi 10.2144/000114292
Vol. 58 | No. 5 | 2015 269
www.BioTechniques.com
suggested true positive by qualitative
Crossing Points
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