Appendix 8.A Illustration of Screening Cut Point Evaluation

Assay response data from 51 disease-matched subject samples in duplicate were available from six runs (two analysts, three runs each). A balanced experimental design (Table 8. A1) was used for this evaluation. Since areportable result for a subject sample during the bioanalysis phase is the average of the response from duplicate samples, all analyses for these validation data were carried out on the average of the duplicate samples. 

The process outlined in Fig. 8.2 was used in the determination of the screening cut point. First, the distribution of the assay response data was assessed. Since the data were relatively more symmetric in the log scale than in the original scale, analyses 

Appendix 8.A.2 Evaluation of Cut Point Based on the Validation Data 

Since the distribution of the log-transformed data after removing these outliers satisfied the Shapiro Wilk normality test (Fig. 8. A2b), the cut point determination for these validation data was made using the parametric method (i.e., mean + 1.645 x SD). The threshold 1.645 for normally distributed data ensures 5% false-positive rate. This cut point of the log-transformed values of the sample replicate means was determined for each analyst, each assay run, and for all the data combined (Table 8.A2). 

Appendix 8.A.3 Comparison of Mean and Variances Across Assay Runs and Selection of Appropriate Type of Screening Cut Point 

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