Parameter | Definition | Experimental details | Sample type | Technical replicates | Deviation from Andreasson et al |
---|---|---|---|---|---|
Robustness | The ability of the assay to accurately and precisely measure the analyte under small protocol variations [23]. | Modify the incubation duration of capture antibody (1 h vs. 2 h vs. overnight), sample (2 h vs. 1 h vs. overnight), and detection antibody (1 h vs. 30 min) parallel assays. Analyze %CV and %recovery. | Plasma pools (low GFAP: 3–6-month-old wildtype mice, intermediate GFAP: 9–24-month-old APP/PS1 mice and 18–24-month-old wildtype littermates, high GFAP: 6 h post-TBI). | 2 | No deviations. |
Precision | The variation in analyte measurement within a single assay (repeatability, intra-assay variation) and between independent assays performed on different days (intermediate precision, inter-assay variation) [23]. | Repeat the assay with the same specimens on 5 independent days. Analyze intra-assay and inter-assay %CV. | Plasma pools (low GFAP: 3–6-month-old wildtype mice, intermediate GFAP: 9–24-month-old APP/PS1 mice and 18–24-month-old wildtype littermates, high GFAP: 6 h post-TBI). | 5 | No deviations. |
Limits of quantification | The lowest and highest analyte concentrations in plasma that can be measured by the assay with a given level of precision (< 20% CV) [23]. | LLOD: Assay replicates of diluent, calculate LLOD as 2.5 SD above the blank. | Diluent (1% BSA in PBS). | 16 | No recommendation available in Andreasson et al [23], followed Meso Scale Discovery recommendations. |
LLOQ: Assay replicates of diluent, calculate LLOQ as 10 SD above the blank. | Diluent (1% BSA in PBS) | 16 | No deviations. | ||
LLOQ: Assay specimens with very low GFAP concentrations. LLOQ = lowest measured concentration with duplicates < 20%CV. | Plasma specimens with low expected concentrations (n = 8, 3–6-month-old wildtype mice). | 2 | No deviations. | ||
ULOQ: Assay specimens with high GFAP concentrations. ULOQ = highest measured concentration with duplicates < 20%CV. | Plasma specimens with high expected concentrations (n = 8, 6 h post-TBI). | 2 | No deviations. | ||
Dilution linearity | The ability of the assay to accurately and reliably detect the analyte in plasma spiked with the calibrator at a very high concentration after dilution [23]. The ability of endogenous analyte in plasma to be detected at various dilutions accurately and reliably [28]. | Spike plasma specimens with recombinant protein 120-fold above the estimated ULOQ (2,000,000 pg/mL), perform serial dilutions until theoretical concentration is below LLOQ (1-fold to 1,000,000-fold). Analyze %CV and %recovery. | Plasma specimens with low expected concentrations (n = 3, 3–6-month-old wildtype mice). | 2 | No deviations. |
Parallelism | Comparison of the signal vs. dilution factor response of the calibrator and endogenous analyte in plasma [23]. | Perform serial dilutions (1-fold to 64-fold) of specimens with high expected concentration. Analyze %CV and %recovery. | Plasma specimens with high expected concentrations (n = 3, 6 h post-TBI). | 2 | 3 specimens were used rather than the recommended 4 specimens, no other deviations. |
Perform serial dilutions (1-fold to 64-fold) of specimens with high expected concentration, analyze response of signal to dilution factor compared to response of recombinant protein. | Definition from Sweeney et al [28]. 3 specimens were used rather than the recommended 5 specimens, no other deviations. | ||||
Recovery | Ability to accurately and reliably measure the concentration of plasma spiked with calibrator [23]. | Divide specimens into 4 aliquots, spike 3 of the aliquots with recombinant protein at concentrations across the range of the standard curve (100, 1000, and 10,000 pg/mL), add an equivalent volume of diluent to the fourth aliquot. Analyze %CV and %recovery. | Plasma specimens with low (n = 2, 3–6-month-old wildtype mice) and high (n = 3, 6 h post-TBI) concentrations. | 1 | No deviations. |
Stability | The stability of the analyte in plasma after a given number of freeze-thaw cycles or a given amount of time at a given temperature [23]. | Divide specimens into 19 aliquots. Aliquot 1 store at − 80 °C. Aliquots 2–6 exposed to 1, 2, 3, 5, or 7 freeze-thaw cycles (2 h at RT then store at − 80 °C), respectively. Aliquots 7–12 store at RT for 1, 2, 4, 24, 72, and 168 h, respectively. Aliquots 13–18 store at 4 °C as above. Aliquot 19 store at −20 °C for 1 month. Assay together and analyze %CV and %recovery. | Plasma pools (low: 3–6 month- old wildtype mice, intermediate: 9–24-month-old APP/PS1 mice and 18–24-month-old wildtype littermates, high: 6 h post-TBI). | 2 | No deviations. |
Selectivity | The ability to measure the analyte in the presence of other substances expected to be present [23]. | A) Compare assay performance in plasma and serum. B) Measure the analyte in specimens with hemolysis. | A) Plasma and serum from sham (n = 6) or TBI (n = 7) mice collected 6 h post-TBI. B) 5%, 25%, or 50% red blood cells spiked into plasma from sham (n = 3) or TBI (n = 3) mice collected 6 h post-TBI. Spiked specimens were frozen at − 80 °C then thawed and assayed. | 2 | Andreasson et al recommends spiking in substances with similar physiochemical structure. |
Pre-analytical factors | The stability of the analyte with respect to blood collection and processing methods. | Blood was collected by saphenous vein and cardiac puncture. Blood from cardiac puncture was divided into 2 aliquots. One aliquot was centrifuged to plasma < 1 h after collection and one aliquot was incubated on ice 4 h before centrifugation. | Plasma from sham (n = 3) or TBI (n = 3) mice collected 6 h post-TBI. | 2 | No recommendation available in Andreasson et al [23]. |