Customisation and validation of a low-volume plasma renin activity immunoassay: Enabling of regulatory compliant determination in paediatric trials

Figures

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  Fig. 1. The modified plasma renin activity assay procedure. PMSF: phenylmethylsulfonyl fluoride; AB: antibody; HRP: horseradish peroxidase; TMB: tetramethylbenzidine; Rinsing: automatic washing step with 5 × 500 μL wash buffer and subsequent residual removal.

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  Fig. 2. The relative error of all conducted calibration standards expressed as boxplots (12 runs). Solid lines: acceptance limits regarding U.S. Food and Drug Administration (FDA) at LLOQ; Dashed line: acceptance limits regarding FDA for the other calibrators. Box: 25–75%; Image 1: 1.5 interquartile range; Image 2: median; Image 3: mean.

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  Fig. 3. The relative error of between-run accuracy (A) and the coefficient of variation (CV) of between-run precision (B) for the plasma renin activity assay. Five individual quality control samples (including the Lower Limit of Quantification (LLOQ) were determined over six runs in quintuplicate. The six determined mean angiotensin I concentration levels for each of the six runs are shown as boxplots for every individual quality control level. Solid lines: acceptance limits regarding U.S. Food and Drug Administration (FDA) at LLOQ; Dashed line: acceptance limits regarding FDA for the other calibrators. Box: 25–75%; Image 1: 1.5 interquartile range; Image 2: median; Image 3: mean.

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  Fig. 4. The percentage deviation of long-term stability for seven sources (indicated by the different symbols). Reference values of plasma renin activity were obtained by calculating the mean of three replicates of each source. Grey dashed lines indicate the maximum accepted deviation regarding U.S. Food and Drug Administration guidelines. Vertical dotted line marks the excluded run due to a processing error.

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  Fig. 5. Youden plot for the results of interlaboratory quality assessment by the Reference Institute for Bioanalytics (Bonn, Germany). The x-axis represents sample A concentration while the y-axis represents sample B concentration. The marked circle indicates the reported values for sample A and sample B. Grey circles indicate other determination methods (radioimmunoassays). The rectangle indicates the maximum limits for a valid result.

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  Fig. 6. The plasma renin activity values (connected points/left y-axis) of three infants with congenital heart diseases before and 4 h after administration of enalapril maleate, as well as the relative deviation of each corresponding incurred sample reanalysis (ISR) (bars/right y-axis). Subject 1: 0.69 years of age; Subject 2: 0.12 years of age; Subject 3: 0.37 years of age; Dashed line: maximum accepted deviation for ISR.