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The overall mean assay quantitation value for seven HCV-positive WHO-standardized Acrometrix NAP linearity panel members was within 0.06 log IU/ml of the mean assigned value.
While rigorous statistical methods, such as mixed effects regression or survival analysis techniques, are the preferred method for deriving mean assay window periods (see e.g., [22]), such methods require substantially larger sample sizes than were available for this analysis.
Instead, we used an approximate method to estimate assay window periods: we observed in the pattern of antibody kinetics the point at which each subject crossed the assay cutoff (0.8 for BED, 0.85 for Ax-AI (Ax-AI cutoff based on personal communication with B. Suligoi)), and then averaged the values across subjects to obtain the mean assay window period.
The reported assay is based on the mean assay of ten individual vials.
The mean assay value in this sample was considerably lower (3.86 ± 2.7) with values ranging from 0.13 to 7.65.
Assay chemical combinations were defined as active in the primary screening if they met a predefined threshold of the mean assay signal differing by at least 30% from the vehicle (DMSO) control signal or the mean assay signal varying by a minimum of 2.0 median absolute deviations from the median (MAD2).
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The mean intra assay coefficient of variance for the three samples was 4.71% range (1.10% to 9.49%) and the mean inter assay coefficient of variance was 4.74% range (1.20% to 9.27%).
Lack of validation does not necessarily mean an assay is invalid.
The mean inter-assay variability was 9.2% and mean intra-assay variability was 11.8%.
In general, a mean intra-assay variation of 10 20% and a mean inter-assay variation of 15 30% on a molecular basis (a maximum variation of 2 and 4% respectively, based on Ct) is realistic over the wide dynamic range.
The mean intra-assay coefficient of variation (CV) for all samples was 8%, and the inter-assay CV for the control plasma was 13% (n = 94 plates).
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