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6.2 Assay validation

While there are multiple methods for assay validation that can be used based on the eventual 685 application, the approach described here and in Table 2 presents a summary of best practices for 686 PCR assay validation for clinical use. Assay validation requires a comprehensive analysis of the 687 test system (i.e. the sample to answer process including all steps such as extraction, testing, and 688 analysis) to define the assay performance characteristics in the intended matrix, and the test 689 system is only validated for that specific use. Both quantitative and qualitative assays require 690 analytical sensitivity and specificity, precision, and accuracy testing with reproducibility via 691 multiple replicates and testing runs being built into the testing design. 692 One comprehensive way of assessing analytical sensitivity in the intended matrix (28) is to 693 conduct a preliminary LOD analysis with a confirmation of LOD, for example, incorporating at 694 least 5 independent samples being run each day over five days. Assay precision reflects the 695 repeatability of an assay when testing multiple aliquots of a sample (29). Validation expands the 696 analytical specificity testing to include an assessment of interfering substances (e.g., heme from 697 whole blood, humic substance from soil samples) on assay performance. Comparing assay 698 linearity and LOD in the intended matrix to a simple matrix can help define that impact. 699 Reproducibility precision testing should incorporate as many potential variables as possible 700 (e.g., different users, days, extraction instruments, thermocyclers, etc.). Based on Clinical and 701 Laboratory Standards Institute (CLSI) guidance (30), precision should involve testing three 702 independent samples (at the LOD and 20 % above and below the LOD) each day for 10 days. 703 Accuracy defines how close the test result is to the actual value (31). In cases where a gold 704 standard comparator is available, a direct comparison to the test assay can be made. In cases 705 where such a comparator is not available, a mock clinical trial or recovery study can be 706

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