Abstract
ObjectivesInvestigations of plasma renin activity (PRA) in children are urgently required. Small-volume, regulatory guideline compliant, bioanalytical assays tailored for paediatric application could facilitate to overcome this hurdle. Ethical constraints given e.g. by the European Medicines Agency need to be addressed and reliable data generation in line with Good Clinical Laboratory Practice must be ensured. MethodsA PRA enzyme-linked immunosorbent assay (ELISA) was tailored for paediatric application and validated in the context of the U.S. Food and Drug Administration bioanalytical guideline. Performance verification of the assay was conducted by participation in an interlaboratory ring test, evaluation of incurred sample reanalysis and an application-orientated approach in children. ResultsA five-fold reduction of required plasma volume to 100 μL was achieved without limiting the calibration range. Between-run accuracy and precision varied no more than 5.0% and 6.3%, respectively. No substantial matrix effect was detected and the inter-run precision for parallelism was 11.1%. Stability experiments approved the freeze-thaw stability, short-term stability as well as 37 weeks of long-term stability. The assay successfully participated in the interlaboratory ring test, showing non-inferiority regarding radioimmunoassay (RIA). Moreover, PRA in plasma samples of neonates was successfully determined. Conducted incurred sample reanalysis confirmed the comparability and reliability of the assay with regard to international regulatory bioanalytical guidelines. ConclusionA fit-for-purpose PRA ELISA characterised by low-volume application was successfully established, indicating non-inferiority regarding commonly applied RIAs. Reliability of the regulatory-compliant PRA assay was proven by participation in an interlaboratory ring test and its application in a paediatric population.
Highlights
Cardiovascular diseases (CVDs) are a leading cause of death in the Western world [1]
The renin-angiotensin-aldosterone system (RAAS) is characterised by an enzymatic cascade, beginning with the precursor peptide angiotensinogen, which is cleaved by the endopeptidase renin to angiotensin I (AngI)
AngI is processed via the angiotensin-converting enzyme (ACE) to the bioactive peptide angiotensin II and corresponding degradation products [5]
Summary
Cardiovascular diseases (CVDs) are a leading cause of death in the Western world [1]. Angiotensin-converting enzyme Angiotensin I Coefficient of variation Cardiovascular disease Calibration standard Enzyme-linked immunosorbent assay European Medicines Agency Food and Drug Administration Good Clinical Laboratory Practice Heart failure Horse radish peroxidase Ligand binding assay Lower Limit of Quantification Optical density Plasma renin activity Pharmacodynamic Phenylmethylsulfonyl fluoride Pharmacokinetic Quality control Renin-angiotensin-aldosterone system Relative error Radioimmunoassay Room temperature Standard deviation Tetramethylbenzidine Upper Limit of Quantification respiratory distress, fatigue and weakness as clinical symptoms [3]. It had been shown that the renin-angiotensin-aldosterone system (RAAS) is actively involved in the development and prognosis of HF [4]. Among the humoral parameters involved in the RAAS, plasma renin activity (PRA) is commonly utilised for evaluating metabolic dysfunctions (e.g. PRA-aldosterone ratio for the diagnosis of primary hyperaldosteronism, a cause of hypertension and trigger for diverse CVDs, including HF) [6]. High rates of off-label and unlicensed drug use in the paediatric population indicate a gap in current knowledge of PRA and further RAAS-related parameters in the very youngest [8]
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