BackgroundPlasma renin levels were determined in the academia-driven, EU-funded “Labeling of Enalapril from Neonates up to Adolescents” (LENA) project to evaluate its role in pediatric heart failure. Quality-controlled bioanalysis is crucial to ensure reliable data generation. However, a comprehensive bioanalytical quality control (QC) concept to monitor the method performance within an academic environment was lacking.MethodsThus, a QC concept was designed encompassing regulatory guidance, international recommendations and current scientific discussions. The concept included (1) a system-suitability test, (2) verification of single bioanalytical runs by calibration curve performance and evaluation of QCs, (3) assessment of the inter-run accuracy according to Clinical Laboratory Standards Institute (CLSI) guideline, (4) monitoring of reproducibility by pediatric incurred samples, (5) blank-sample analysis and (6) participation in interlaboratory testing.ResultsThe concept was successfully applied to the academic project. About 11% of single runs were identified as invalid and triggered a re-analysis of unknown samples being included in those runs. The usefulness of the customized inter-run monitoring was demonstrated and proved the good accuracy from the first to the last run. All 147 reanalyzed incurred sample pairs complied with regulatory requirements.ConclusionsThe regulatory complied QC concept was customized for the demands of academia-driven pediatric trials and contributed to the reliable quantification of 965 pediatric renin samples.
As part of the extended renin-angiotensin-aldosterone system, active renin and its precursor prorenin have been an area of research interest for decades. Although several studies showed a correlation with disease, other studies found no significant association, e.g. attributed to limited sample size or pharmacological effects of antihypertensive drugs. Since the measurement of both proteins has typically been carried out in adult populations, the data in paediatrics is limited. This review aimed to collate the current data on plasma renin and prorenin levels in children and compare the levels of healthy vs. the diseased state. A literature search using Medline resulted in 213 publications of which 15 studies were classified as relevant. In the extant studies in the literature, an age-dependent decline of renin plasma concentration was observed in newborns compared to adolescents. For children with cardiovascular disease, five studies were identified that provide limited insight into the pathophysiological regulation of renin. In general, sample handling is still a crucial step, which might particularly affect measured active renin concentrations due to conformational changes of its precursor prorenin. A reliable assessment for prorenin levels in the maturating population is yet not possible due to the low number of available publications. Three different approaches to quantify prorenin were found and raise the question on the comparability of these methods. The review emphazised several weaknesses and highlights the need for an accurate procedure to determine levels of active renin as well as prorenin in its closed and open form.
Rationale Human prorenin, representing the precursor of mature renin, has been discussed as a potential biomarker, e.g. in diagnosing primary hyperaldosteronism or diabetes‐induced nephropathy. Currently, only immunoassays are available for prorenin quantification. As the similarity of prorenin to active renin impedes its accurate determination by immunoassay, mass spectrometry appears as an accurate alternative for differentiation of that protein. Methods Immunoaffinity purification plus a mixed‐solvent‐triggered digestion was combined with liquid chromatography/mass spectrometry (LC/MS) to enable a fast, sensitive, and less laboratory‐intensive approach to the quantification of prorenin. Statistical experimental planning, which is known as Design of Experiments (DOE), was used to identify the optimal conditions for the generation of the signature peptides within a manageable number of experiments. The efficiency of the mixed‐solvent‐triggered digestion by trypsin was investigated using four different organic solvents: acetonitrile, acetone, tetrahydrofuran and methanol. Results By utilizing a D‐optimal design, we found that the optimal mixed‐solvent type for the generation of both signature peptides was acetonitrile at a concentration of 84% and an incubation temperature of 16°C. Using the mixed‐solvent‐triggered digestion, the procedure time allowed a fast analysis of active renin and prorenin with a short digestion time of 98 min. This optimized mixed‐solvent‐triggered digestion procedure was applied to detect renin and prorenin successfully in human plasma by the newly developed hybrid approach. Conclusions The identification of unique surrogates for human prorenin enabled the mass spectrometric differentiation between the two similar proteins. The novel hybrid approach successfully proved its ability to purify, detect and distinguish between prorenin and active renin in human plasma.
BackgroundAs the initiator of the Renin-Angiotensin-Aldosterone-system, renin plays an essential role in the vicious circle of heart failure. Therefore, renin was determined in the investigators driven ‘Labelling of Enalapril from neonates up to adolescents’ (LENA) study to evaluate its role in paediatric heart failure. Due to the often long-lasting periods of recruitment of paediatric subjects, the assay performance has to be guaranteed over the whole recruiting time. Therefore, to ensure the high quality of the determined renin study samples after successful assay validation,1 a multi-step quality approach was used to get reliable results over a period of 30 months.MethodsBased on a multi-step quality approach consisting of calibration standards (CSs), quality controls (QCs) and incurred sample reanalysis (ISR), study samples of unknown renin concentrations were determined. Results within predefined limits of CSs (6 levels) and QCs according to European Medicine Agency (EMA) guidelines were required for evaluating the study samples.2 ISR was performed for randomly selected paediatric samples to evaluate the long-term accuracy of the validated assay.Results133 analytical runs were conducted for renin from February 2016 to August 2018. In 119 (88.8%) valid runs, a total number of 1414 of CCs and 952 of QCs were determined. Thereof 99.9% of CCs and 98.3% of QCs were in the predefined limits according to EMA. 143 incurred sample pairs were reanalysed resulting in 95.8% of samples within EMA guidelines. Using this multi-step quality approach, the reliable determination of 965 LENA paediatric study samples was guaranteed.ConclusionIn addition to the assay validation, the multi-step quality approach ensured the reliability of the determined renin concentrations in the continuous bioanalysis of the paediatric study samples and guaranteed the high quality of the collected data in the LENA study.ReferencesSchaefer J, Burckhardt BB, Tins J, et al. Validated low-volume immunoassay for the reliable determination of direct renin especially valuable for pediatric investigations. J Immunoass Immunochem 2017;38:579–94. doi:10.1080/15321819.2017.1350707Guideline on bioanalytical method validation. European Medicines Agency, London, UK (2011).Disclosure(s)Martin Feickert, Ilja Burdman, Nina Makowski, Moshin Ali, Anke Bartel, and Bjoern B. Burckhardt declare that there is no conflict of interest. The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007–2013) under grant agreement n°602295 (LENA)
BackgroundSince sample volume is limited in children, innovative bioanalytical methods and enrichment procedures are highly required. The analysis of endogenous substances by liquid chromatography coupled to mass spectrometry is a highly specific method because of its selectivity and accuracy. However reliable detection of endogenous substances can only be achieved by a hybrid assay approach combining immunocapture and mass spectrometry. Key element of the immunocapture procedure is the selection of the appropriate antibody for capturing the desired antigen. This study is meant to identify the most suitable antibody that facilitates the development of an hybrid assay approach concerning reliable detection of endogenous prorenin in paediatric samples.MethodsDynabeads magnetic beads were coupled to three different antibodies from three different vendors (GeneTex, Molecular Innovations, R&D systems). 500 µL human plasma which was spiked with 20 ng recombinant human prorenin (Cayman chemicals). The immunocapture step was followed by protease digestion and a custom-made µelution solid-phase extraction protocol. The digest was analyzed by Shimadzu Nexera LC-system coupled with Sciex TripleTOF 6600 mass spectrometer.ResultsThe analysis of the captured prorenin was performed by the surrogate peptide approach. In this case the surrogate peptide was identified as unique. The comparison of the three available antibodies showed that one antibody did not ensure reliable binding properties in human matrix. Among the two remaining antibodies only one showed sufficient binding capacities to be applied in small sample volumes commonly available in paediatric samples. Using this hybrid approach enabled the enrichment of the required volume by factor of 20.ConclusionThis study identified the most suitable antibody for the immunocapture procedure of the prorenin hybrid approach. This is now followed by further mass spectrometric method development and validation prior to its application in paediatric samples.Disclosure(s)Declaration of interest: none Ilja Burdman and Bjoern B. Burckhardt declare that there is no conflict of interest. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors
BackgroundThe physiological and pathophysiological characteristics regarding plasma renin activity (PRA) in the context of the renin-angiotensin-aldosterone-system (RAAS) are well investigated in adults, whereas less is known in paediatric population suffering from heart failure. Challenges in the conduct of paediatric investigations limit the enrolled children often to specific age groups allowing only a partial revaluation of the maturating RAAS. To constitute the comprehensive picture of the paediatric RAAS from 0–18 years, a compilation of available PRA data was conducted via literature search.MethodsA systematic literature search was accomplished in the context of the ‘Labeling of Enalapril from Neonates up to Adolescence’ (LENA) project in the MEDLINE database via PubMed in January 2019. Key words: plasma renin activity and congenital heart disease/dilative cardiomyopathy/heart failure/congenital heart defect and child/neonate/infant/toddler/paediatric. Eligible records included PRA values in children of 0–18 years of age. Exclusion criteria comprised foetuses, preterm, cord blood, urine, adults, < 2500 g birthweight, ex vivo studies and deviant diseases.ResultsLiterature search resulted in 167 findings of which 58 full-text articles were assessed for eligibility. Finally, 33 publications were classified as relevant. Of these, 21 and 12 records were assigned for healthy and cardiac diseased population respectively, leading to PRA data sets of 2000 healthy and 254 diseased children. Visual check of data revealed an age dependent decrease of PRA, in particular in the early childhood, and a substantial elevation of PRA in heart failure patients.ConclusionThe conducted literature search allowed a systematic description of PRA values in healthy and cardiac diseased paediatrics, which facilitates a classification of reference ranges of the maturing RAAS for LENA and future paediatric trials.Disclosure(s)Fabian K. Suessenbach, Tanja Gangnus, Ilja Burdman, Nina Makowski, Stephanie Laeer and Bjoern B Burckhardt declare that there is no conflict of interest. The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007–2013) under grant agreement n°602295 (LENA).
BackgroundN-terminal pro-brain natriuretic peptide (NT-proBNP) is a valuable biomarker for diagnosis and prognosis of heart failure in adults, included into the European Society Guidelines for heart failure (2016).1 It is also considered as a diagnostic and follow-up marker in paediatric heart failure. The aetiology of paediatric heart failure is heterogeneous and maturation of the cardiac and neurohumoral system influences NT-proBNP levels. Since substantial information is mandatory to enable a long-term follow-up of children with heart failure, the aim was to collect published paediatric NT-proBNP data.MethodsIn January 2019, a literature search using PubMed was performed comprising the following keywords: NT-proBNP, heart failure/dilated cardiomyopathy/congenital heart defect/congenital heart disease/healthy and child/neonate/toddler/infant/paediatric. Eligible publications had to determine levels of NT-proBNP in plasma or serum in paediatric heart failure or healthy children (0–18 years) with the Roche NT-proBNP-immunoassay.ResultsThe search resulted in 343 records, of which 95 measured NT-proBNP in paediatric controls or heart failure. Of them, 48 studies were excluded due to the use of other immunoassays. Following, 47 studies were included into the analysis of which 27 reported NT-proBNP levels in 3435 healthy children and 38 NT-proBNP concentrations in 1885 children with heart failure. The age range of reported levels comprised the day of birth up to 18 years in both groups. The data set revealed that younger children have higher NT-proBNP values than older children and that heart failure patients had increased NT-proBNP levels compared to healthy controls which are also dependent on the severity of disease.ConclusionThe literature search and analysis confirmed that NT-proBNP is an important marker for the detection of heart failure and classification of disease severity in children. Thus, the compiled data set forms a solid data basis for long-term follow-up of a paediatric patient population with heart failure.ReferencesPonikowski P, Voors AA, Anker SD, et al. 2016 ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure: The task force for the diagnosis and treatment of acute and chronic heart failure of the european society of cardiology (ESC)developed with the special contribution of the heart failure association (HFA) of the esc. Eur Heart J 2016;37(27):2129–200.Disclosure(s)Tanja Gangnus, Fabian K. Suessenbach, Nina Makowski, Ilja Burdman, Stephanie Laeer, Björn B. Burckhardt declare that there is no conflict of interest. The research leading to these results has received funding from the European Union Seventh Framework programme (FP/2007–2013) under grant agreement n°602295 (LENA).
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