SPADA Draft Documents

qualitative assays require analytical sensitivity and specificity, precision, and accuracy testing 657 with reproducibility being built into the testing design. 658 Analytical sensitivity in the intended matrix (26) is identified by conducting a preliminary 659 LOD with a confirmation of LOD incorporating at least 5 independent samples being run each 660 day over five days. Assay precision reflects the repeatability of an assay when repeatedly testing 661 multiple aliquots of the sample (27). Validation expands the analytical specificity testing to 662 include an assessment of interfering substances (e.g., heme from whole blood, and humic 663 substance from soil samples) on assay performance. Comparing assay linearity and LOD in the 664 intended matrix to a simple matrix can help define that impact. 665 Reproducibility precision testing should incorporate all the potential variables when running 666 the test (e.g., different users, days, extraction instruments, thermocyclers, etc.). Based on CLSI 667 guidance (28), precision should involve testing three independent samples (at the LOD and 20% 668 above and below the LOD) each day for 10 days. Accuracy defines how close the test result is to 669 the actual value (29). In cases where a gold standard comparator is available, a direct comparison 670 to the test assay can be made. In cases where such a comparator is not available, a mock clinical 671 trial or recovery study can be performed (30). Here, 50 positive samples (1/3 at 1.5x LOD, 1/3 at 672 a mid-relevant range, and 1/3 at a high relevant range) and 100 negative samples are tested over 673 five days (31). Precision testing for quantitative assays is similar to qualitative assays except the 674 samples generated are at a high concentration, a low concentration, and at the assay LOD (32). 675 In addition to the above analyses, quantitative assays require defining the reportable range of 676 the assay, that is, the linear range of the standard curve where quantitative results can be 677 accurately reported (33). Defining the lower limit of quantification (LLOQ) is done by testing 30 678 independent samples in duplicate at the lowest concentration within the reportable range and 679

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