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A stepwise regression was performed in each replicated sample with thresholds of p = 0.05 for variable selection and for variable elimination [ 19].
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Five thousand lymphocyte events are acquired per sample with threshold for acquisition set on the FL1 (FITC) channel.
With the Panleucogate gating strategy (9, 10), 5,000 lymphocytes are acquired per sample with threshold for acquisition set on the forward scatter (size) channel.
Samples with threshold cycle (C T) values below 40 were considered positive (28).
$$\end{document} Samples with a threshold cycle higher than the chosen cutoff point are classified as negative, whereas samples with threshold cycle lower than the cutoff point are classified as positive [ 30].
Samples with threshold cycle (Ct) values ≥ 35 or those where the standard deviation across a triplicate was ≥ 0.5 were considered failed reactions and re-run.
Test samples with threshold cycle numbers that fell outside the range defined by the standard curves were rerun at different concentrations to ensure that they were amplified within the linear range.
Any sample with a threshold cycle (CT) of <35 was considered to be positive for WNV.
People who refused to participate in the study could benefit from routine screening (one sample with positivity threshold at 100 ng ml−1).
We used the cumulative matching curve (CMC) for identification and the receiver operating characteristic curve with z-normalization (z-ROC), which indicates the trade-off between the false rejection rate (FRR) of genuine samples and false acceptance rate (FAR) of imposter samples with varying thresholds for verification.
Samples with cycle thresholds for detection (Cts) below 35 and undetectable results from the RT negative control were deemed evaluable for use in a Taqman array.
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