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All samples received a vacuum heat treatment and were subsequently subjected to thermal cycling in air.
At the beginning of data processing all samples received a unique three digit number, and during further analyses this number was used without referring to the patient's name.
Three samples received a code of 3 and are not included in results here.
All samples received a unique identifier number and demographic, medical and genetic data were stored in a centralized database.
All samples received a score for the intensity of the immunohistochemical staining, percent of the tissue core stained, and an IR score that is the product of the staining intensity and percent stained [ 5].
In particular, only 42% of the samples received a concordant class assignment, while the ER status, HER2 status, pathological grading and tumor size were all correlated with the number of times a sample was classified as poor outcome by the signatures.
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Control samples received an ID injection of 10µl of sterile PBS.
Overall, 6 of the 11 tissue types had a classification accuracy above 92 %, though 10.4%% of all samples received an Undetermined classification.
The protocols adopted in this study yielded four different plasma samples: each was subjected to a standard procedure, but three samples received an additional spin of either increasing duration or centrifugal force (see Methods and Figure 4).
In addition, the methodology was validated with real serum samples, receiving a good correlation with the results obtained from commercially available electrochemiluminescence automated analyzer.
In addition, the methodology was also validated by assaying 16 positive serum samples and 7 negative serum samples, receiving a good correlation with the results obtained from the referenced electrochemiluminescence method.
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