Parameter | Criteria | Achieved | Data |
Precision and accuracy measuring calibrators | 75% of calibrators CV≤20% and 75% of calibrators DFT≤±20%. | 1×assay 89%. 6× assays 100% of calibrators CV≤20%. 1×assay 89%. 6×assays 100% of calibrators DFT ≤±20%. | Figure 3A and B. online supplemental table 1 and 2. |
Precision and accuracy measuring QC samples | High (140 pg/mL), medium (75 pg/mL) and low (15 pg/mL) QCs CV ≤20% and DFT≤±20%. | 6/7 assays all QCs had CV≤20%. 6/7 assays all QCs had DFT≤±20%. | Figure 3C and E. online supplemental table 3 and 4. |
Intraplate and interplate reproducibility | Repeat measure of QC samples across multiple plates and positioned across a single plate CV ≤20%. Three sets QC samples prepared independently, in two independent assays by two analysts, CV ≤20% and DFT≤±20%. | 100% of repeat measures of QC samples CV ≤20%. 100% of QC sets, prepared by two analysts CV≤20% and DFT≤±20%. | Figure 3D and F. online supplemental table 5 and 6. |
Precision measuring matrix control sample | Repeated measures of a positive human C9orf72 CSF sample should have CV≤20%. | Raw AEB and predicted GP concentration from four assays CV≤20%. | Figure 3G. online supplemental table 7. |
Dilutional parallelism | At least three of diluted samples within the assay’s range should have DFT within ±30.0% | Using 1:2 as anchor, 4/6 samples at 1:4 had DFT within ±30.0% | Figure 3H. online supplemental table 8 and figure 1. |
Freeze–thaw stability | Freeze–thaw stability of matrix control QC. CV ≤25% and DFT ≤±30%. Freeze–thaw stability of calibrators CV≤20%. | After three Freeze–thaw cycles matrix control QC CV≤25% and DFT≤±30%. After three freeze–thaw cycles of calibrators 100% CV ≤20%. | Figure 3. online supplemental table 8 and 10. |
Haemoglobin tolerance | Assay should tolerate low levels of haemoglobin within ±20%. | Assay tolerates 0.2% haemolysate spike with measures within ±20%. | Figure 3I and J. online supplemental figure 2. |
AEB, average number of enzyme labels per bead.