Clustering of TAC results in the FCN-MR plane.

<p>A: output of the MR method for the training dataset. Reactions were identified by a consensus classification as either positive (●) or negative (■) and are plotted in the FCN-MR plane, showing the different localization of the two populations. Since early cycles of amplification might be biased by transient in fluorescence that can generate high MR values, two MR cut-offs were applied to separate positive and negative results. For values below the lowest FCN of the positive population (9.47, solid line), 150% of the maximum MR observed in the region was used (0.068, dotted line). Histogram analysis was implemented to obtain MR values that could separate the positive and negative populations (insert). The mid-point between the lowest and highest of these separators was used to discriminate positive and negative results for reactions with a FCN values above the lowest FCN of the positive population (0.018, dashed line). B: the cut-off values empirically obtained by analysing the training dataset were applied to the query dataset in order to identify positive and negative reactions. The results are classifies as true positive (●), true negative (■), false positive (▲) and false negative (×) according to the consensus classification. The FCN axis has units defined in cycles whereas the MR axis is a-dimensional. The x-axis of the insert panel is given in MR values.</p

Representative amplification profile of the MR false positive results.

<p>Fluorescence amplification profile of the single reaction identified as false positive by the MR method in comparison to the consensus classification. A: the normalized fluorescence profiles indicates a proper amplification but the signal is weak, being only about three times the cut-off level of 0.2 fluorescence units (horizontal line). The Cq of this reaction (32.42 cycles) is reported (vertical line). B: the MR response for this reaction shows a good profile but the MR value (horizontal line) is below the empirical cut-off obtained using the training dataset, therefore the reaction was identified as negative. The FCN value (33.82 cycles) is depicted (vertical line).</p

Examples of amplification profiles and MR responses.

<p>Representative amplification profiles of typical cases observed in this study. Sigmoid profile derived from a clear positive sample observed with the CT method (1) and the associated Gaussian profile of the MR response (4). Low fluorescence signal that crossed the threshold level giving a positive result with a low Cq value using the CT (2) but not with the MR (5) method. Transient peaks in fluorescence resulted in high Cq value with the CT method (3) but gave a negative result using the MR method (6). The y-axis is provided in different scales across the panels.</p

Agreement between mathematical methods and raters in sample subsets.

<p>The Fleiss' κ values obtained for selected subsets are stratified according to the raters one another or the raters together with either the CT or MR methods. The query subset was obtained by considering as negative the CT results with a Cq above 40 cycles; the other subsets included also Cq above this level to be consistent with the original clinical dataset. The p-values for the Fleiss' κ test were all lower than 0.001. The accuracy was based on the consensus classification. The number of reactions per group is reported (n).</p

Standard curves obtained by CT and MR methods.

<p>The Cq (●) and FCN (▲) values for the different plasmid dilutions are depicted. The regression lines built on these values represent the standard curves for the CT (solid line) and MR (dotted line) methods.</p

Comparison of the CT and MR methods.

<p>The assay parameters for the CT and MR methods are provided for each rater and for the consensus classification. PPV = positive predictive value; NPV = negative predictive value.</p