Table 2

Biomarker assay qualification criteria for poly(GP) single molecule array assay. Coefficient of variation (CV)=(SD / mean)×100. Difference from Total (DFT)=difference from predicted concentration of calibrators (pg/mL from actual, as % of actual. Quality control samples (QCs) were prepared using GST-GP32 in diluent A.

ParameterCriteriaAchievedData
Precision and accuracy measuring calibrators75% 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 samplesHigh (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 reproducibilityRepeat 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 sampleRepeated 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 parallelismAt 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 stabilityFreeze–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 toleranceAssay 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.