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Serum samples would be aliquoted and stored in -80°C at the research laboratory.
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Self-reports of smoking might be prone to measurement error; an objective measure of smoking, such as serum cotinine samples, would be preferable.
Convalescent-phase serum samples would have better defined the cases.
Under this scenario a serum sample would not have been collected during the time that a single initiating autoantibody would have been present.
Since the main pathways related to PF were associated with hemostasis, serum would be a proper biological sample to study the disease.
As serum would be a direct comparison with the calf serum above, for some samples we let the blood clot and took the supernatant (serum) remaining.
The antibody endpoint titer is calculated with Watson 7.4.1 Immune Response Module (Thermo Fisher Scientific, Waltham, MA), and the data are presented as endpoint log titers (log10; endpoint titer is defined as the reciprocal of the serum dilution at which the sample response would be equal to the cut point of the assay).
In DNA extracts from serum samples less host DNA would be present to interfere with the PCR, and inhibiting factors from the blood, such as heme, would also be absent.
Ideally serum samples should be analyzed.
Using pilot data it was shown that in order to detect a two-fold change in peak intensity with a power of 95% at a significance level of α=0.001 (to control false discovery rate) in 90% of the peaks in the serum profile, 68 samples analysed in duplicate would be required in each group.
The estimated annual serum level is equivalent to what would be measured in a serum sample taken during that year, whereas the cumulative measure is the area under the serum level profile curve.
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