Table III.
Parameters for Evaluation During Prevalidation and Validation Experiments, Including Target Acceptance Criteria
| Parameter to be measured | Number of plates for prevalidation | Number of plates for validation | Recommended target values (within quantitative range) |
|---|---|---|---|
| Standard curve accuracy and precision | Two plates | Six to ten plates |
Mean % CV of back-calculated concentrations ≤20% (inter-assay) Mean %RE of back-calculated concentrations ≤20%. Note that precision during validation is tested using validation samples prepared at three to five levels. |
| Validation samples | N/A | Three to five levels (LLOQ, low, mid, high, ULOQ) in chosen matrix | Test in duplicate over 3–5 days with multiple scientists. Results for each analyte should be accurate (%RE ≤30%) and precise (% CV ≤30%). |
| Limit of quantitation | N/A | N/A | Calculated using multiple runs from validation for each analyte |
| Parallelism or endogenous dilutional linearity (if endogenous levels are high enough) | Five individuals, disease-state individuals if available | Ten individuals, disease-state individuals if possible | Results for each dilution when recalculated for the dilution factor should be 100% original (neat) result ± (inter-assay CV% ×3). This is equivalent to accepting ±3SD of the expected result (approximate confidence limit 99.5%). This means that an acceptance limit is statistically significant to the specific method’s performance. |
| Matrix spike recovery | Five individual samples, unspiked and spiked at high QC and low QC levels, using disease-state individuals if available. | Ten individual samples, unspiked and spiked at high QC and low QC levels, using disease-state individuals if available. |
For both low and high spike: 3/5 during prevalidation and 7/10 during validation yield 70–130% of expected concentrations (sum of endogenous level plus spike level); % Recovery = Measured Concentration / Expected Concentration × 100%. |
| Dilutional linearity of high-spiked recombinant protein | Five individual disease-state samples (if available), spiking analyte at high QC levels, and diluted through assay range. | Five individual disease-state samples (if possible), spiking analyte at high QC levels, and diluted through assay range. | Results for each dilution when recalculated for the dilution factor should be within ±3SD of the original (neat) result. |
| Stability | N/A | If possible, three levels of endogenous samples. | ≥24 h at room temperature, ≥5 cycles of freeze/thaw. The mean values for stressed matrix should be within ±3SD of the mean values for unstressed matrix at each level. |
Quality controls are used during sample testing. It is recommended to test three levels chosen based on prevalidation experiments spanning the standard curve. It is expected that results for each analyte should be accurate (%RE ≤30%), and precise (% CV ≤30%) following general 4-6-30 rule; however, criteria may be flexible for certain analytes depending on the intended use, biological variability, etc.
N/A not applicable, % CV percent coefficient variation, %RE percent relative error, LLOQ lower limit of quantitation, ULOQ upper limit of quantitation