Assay validation/verification are essential, for laboratory developed tests (LDTs) and those that are commercially available (FDA approved). Validation of multiplex PCR assays ensures all targets detect equally.
For verification with FDA approved assays, laboratories should perform the essential functions of:
- Accuracy - can you match the manufacturer's claims? Use available calibrators/calibration and quality control material with known values, proficiency testing material with known values and previously tested samples with known values. Generally, 10 positive and ten negative samples suffice to establish accuracy
- Precision - can the lab repeatedly test the same samples on three different days with three different operators (technologists) and get the same or comparable results?
- Reportable range - how high and how low test results can be accurately determined? Generally, select samples with known values at the highest and lowest manufacturer's accuracy claim. Results can only be reported for this range.
Validation for modified FDA assays and LDTs: provides the data required to meet regulatory criteria for implementation. "The analytical validation under CLIA looks at the performance characteristics of a test used to describe the quality of patient test results, and includes an analysis of accuracy, precision, analytical sensitivity, analytical specificity, reportable range, reference interval, and any other performance characteristics required for the test system in the laboratory that intends to use it. This analytical validation is limited to the specific conditions, staff, equipment and patient population of the particular laboratory, so the findings of these laboratory-specific analytical validation are not meaningful outside of the laboratory that did the analysis."
The following assay parameters should be evaluated when validating a multiplex PCR assay, in this order:
- Define targets
- Analytical sensitivity
- Analytical specificity