Assessing the commutability of candidate reference materials for the harmonization of neurofilament light measurements in blood

OBJECTIVES: Neurofilament light chain (NfL) concentration in blood is a biomarker of neuro-axonal injury in the nervous system and there now exist several assays with high enough sensitivity to measure NfL in serum and plasma. There is a need for harmonization with the goal of creating a certified reference material (CRM) for NfL and an early step in such an effort is to determine the best matrix for the CRM. This is done in a commutability study and here the results of the first one for NfL in blood is presented. METHODS: Forty paired individual serum and plasma samples were analyzed for NfL on four different analytical platforms. Neat and differently spiked serum and plasma were evaluated for their suitability as a CRM using the difference in bias approach. RESULTS: The correlation between the different platforms with regards to measured NfL concentrations were very high (Spearman's ρ≥0.96). Samples spiked with cerebrospinal fluid (CSF) showed higher commutability compared to samples spiked with recombinant human NfL protein and serum seems to be a better choice than plasma as the matrix for a CRM. CONCLUSIONS: The results from this first commutability study on NfL in serum/plasma showed that it is feasible to create a CRM for NfL in blood and that spiking should be done using CSF rather than with recombinant human NfL protein

Propulsion de liposomes géants par polymérisation d'actine: un modèle pour l'interaction dynamique cytosquelette-membrane dans la motilité

Cell movement is organized by molecular processes which couple plasma membrane to actin cytoskeleton dynamics, in order to generate cell protrusions. I used a biomimetic approach to reconstitute in vitro site-directed actin polymerization against lipid membranes. To do so, I first synthesised giant unilamellar vesicles (GUVs) by electroformation and functionalized them with actin polymerization activators such as N-WASP. When placed in a reconstituted motility assay made of pure proteins, vesicle grow a branched actin network that propels them in the motility medium. I showed that actin-propelled GUVs undergo continuous or periodic saltatory movement, and that the transition between these two regimes is controlled by the concentration of Arp2/3 complex, which branches filaments by interacting with liposome-bound N-WASP. Phase contrast and fluorescence microscopy show that saltatory motion is linked to cycles in the distribution of N-WASP at the membrane, between a homogeneous and a segregated state. Comparison of the changes in the distributions of N-WASP, Arp2/3 and actin during propulsion demonstrates that actin filaments transiently bind to N-WASP, and that these transient bonds are mediated by the Arp2/3 complex. The interaction of N-WASP-Arp2/3 with the filaments drives segregation. The balance between segregation and free diffusion determines whether continuous movement can be sustained. Computed surface distributions of N-WASP, Arp2/3 complex, and actin, derived from a theoretical description of this mechanism, account satisfactorily for the measured density profiles.Le mouvement cellulaire est organisé par des processus moléculaires qui couplent la dynamique de la membrane plasmique à celle du cytosquelette d'actine, pour engendrer des protrusions cellulaires. Pour analyser le lien fonctionnel entre ces processus et le comportement motile qui en résulte, j'ai utilisé une approche biomimétique. J'ai mis au point une méthode rapide d'électrogonflement de liposomes géants unilamellaires, que j'ai fonctionnalisés avec la protéine N-WASP. Cette protéine catalyse la formation d'un réseau de filaments branchés par le complexe Arp2/3 au bord avant des cellules motiles. Les liposomes, placés dans un milieu reconstitué contenant l'actine, Arp2/3 et les protéines de régulation du treadmilling, sont déformés par la polymérisation insertionnelle de l'actine et se propulsent in vitro. J'ai montré que les liposomes adoptent un régime de propulsion soit continu, soit saltatoire périodique, la transition entre les deux régimes étant contrôlée par la concentration de Arp2/3. Les résultats établissent que le complexe Arp2/3 est le partenaire de N-WASP responsable de l'interaction entre la membrane et les filaments au cours de la réaction de branchement. Cette interaction est transitoire et détermine l'équilibre ségrégation-diffusion de N-WASP dans la bicouche lipidique et la formation d'un réseau cohésif de filaments branchés. Le modèle physique que nous proposons selon lequel l'équilibre ségrégation-diffusion de NWASP est contrôlé par les paramètres cinétiques du cycle catalytique de branchement, reproduit quantitativement les profils de densité de surface de NWASP observés expérimentalement

Propulsion de liposomes géants par polymérisation d'actine (un modèle pour l'interaction dynamique cytosquelette-membrane dans la motilité)

Le mouvement cellulaire est organisé par des processus moléculaires qui couplent la dynamique de la membrane plasmique à celle du cytosquelette d actine, pour engendrer des protrusions cellulaires. Pour analyser le lien fonctionnel entre ces processus et le comportement motile qui en résulte, j ai utilisé une approche biomimétique. J ai mis au point une méthode rapide d électrogonflement de liposomes géants unilamellaires, que j ai fonctionnalisés avec la protéine N-WASP. Cette protéine catalyse la formation d un réseau de filaments branchés par le complexe Arp2/3 au bord avant des cellules motiles. Les liposomes, placés dans un milieu reconstitué contenant l actine, Arp2/3 et les protéines de régulation du treadmilling, sont déformés par la polymérisation insertionnelle de l actine et se propulsent in vitro. J ai montré que les liposomes adoptent un régime de propulsion soit continu, soit saltatoire périodique, la transition entre les deux régimes étant contrôlée par la concentration de Arp2/3. Les résultats établissent que le complexe Arp2/3 est le partenaire de N-WASP responsable de l interaction entre la membrane et les filaments au cours de la réaction de branchement. Cette interaction est transitoire et détermine l équilibre ségrégation-diffusion de N-WASP dans la bicouche lipidique et la formation d un réseau cohésif de filaments branchés. Le modèle physique que nous proposons selon lequel l équilibre ségrégation-diffusion de N-WASP est contrôlé par les paramètres cinétiques du cycle catalytique de branchement, reproduit quantitativement les profils de densité de surface de N-WASP observés expérimentalement.Cell movement is organized by molecular processes which couple plasma membrane to actin cytoskeleton dynamics, in order to generate cell protrusions. I used a biomimetic approach to reconstitute in vitro site-directed actin polymerization against lipid membranes. To do so, I first synthesised giant unilamellar vesicles (GUVs) by electroformation and functionalized them with actin polymerization activators such as N-WASP. When placed in a reconstituted motility assay made of pure proteins, vesicle grow a branched actin network that propels them in the motility medium. I showed that actin-propelled GUVs undergo continuous or periodic saltatory movement, and that the transition between these two regimes is controlled by the concentration of Arp2/3 complex, which branches filaments by interacting with liposome-bound N-WASP. Phase contrast and fluorescence microscopy show that saltatory motion is linked to cycles in the distribution of N-WASP at the membrane, between a homogeneous and a segregated state. Comparison of the changes in the distributions of N-WASP, Arp2/3 and actin during propulsion demonstrates that actin filaments transiently bind to N-WASP, and that these transient bonds are mediated by the Arp2/3 complex. The interaction of N-WASP-Arp2/3 with the filaments drives segregation. The balance between segregation and free diffusion determines whether continuous movement can be sustained. Computed surface distributions of N-WASP, Arp2/3 complex, and actin, derived from a theoretical description of this mechanism, account satisfactorily for the measured density profiles.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Développement de méthodes de référence pour les biomarqueurs du bilan lipidique (application au contrôle qualité en biologie clinique)

En biologie clinique, il est indispensable de disposer de mesures fiables et comparables dans le temps et entre les laboratoires afin de permettre un dépistage et un suivi appropriés des patients. Pour cela, il est indispensable d établir leur traçabilité métrologique aux unités du système international notamment par des méthodes de référence primaires ou des matériaux de référence certifiés (MRC) d ordre supérieur. Ces travaux de thèse ont consisté à développer et valider des méthodes de référence pour le cholestérol total, les triglycérides, le HDL-cholestérol et le LDL-cholestérol. Leur valeur ajoutée par rapport à une valeur consensuelle a été démontrée lors d évaluations externes de la qualité. Elles ont également permis de certifier un MRC qui pourra être utilisé pour le contrôle qualité et/ou l étalonnage des méthodes de routine. Nous avons montré que le MRC était commutable pour la plupart des méthodes de routine pour les différents biomarqueurs, ce qui a permis de l utiliser pour évaluer leur justesse. Les méthodes de routine avaient généralement tendance à sous-estimer la concentration en triglycérides (en particulier aux valeurs basses) et à surestimer nettement la concentration de cholestérol total et de LDL-cholestérol (en particulier aux concentrations proches du seuil de décision clinique), ce qui se traduit par une augmentation du nombre de faux-positifs (patients traités à tort). Une approche de correction de non commutabilité a également été proposée afin de permettre l utilisation de matériaux non commutables pour évaluer la justesse. Pour conclure, ces travaux ont démontré l importance de disposer de méthodes de référence ainsi que de MRC commutablesReliable measurements in medical biology are essential for early screening and appropriate follow-up of patients. Ensuring metrological traceability of clinical measurements to higher order reference methods or certified reference materials enables to obtain comparable results over time and between different laboratories that could use different methods to quantify the same biomarker.In this study, reference methods were developed and validated for lipid profile biomarkers (total cholesterol, triglycerides, HDL-C, and LDL-C). Their value added in proficiency testing schemes was demonstrated against consensus mean. They were also used to characterize a certified reference material (CRM) that may be used both as quality control and/or calibrator of field methods. The CRM was shown to be commutable for most field methods and lipid profile biomarkers, which proved it was suitable to assess trueness. Results of our multicenter study showed that field methods tend to underestimate triglycerides (particularly at low concentrations) and overestimate total cholesterol and LDL-C (especially around the clinical threshold), resulting in false positives and significant patient misclassifications. An approach of non-commutanility correction was also presented to allow trueness assessment with non-commutable samples. In conclusion, this work highlights the importance of using reference methods and also commutable CRM to rigorously assess accuracy of field methods used in clinical laboratoriesDIJON-BU Doc.électronique (212319901) / SudocSudocFranceF

Actualités sur les méthodes de référence pour le dosage des protéines urinaires

International audienceMeasurement of urine albumin has been introduced in the new classification of kidney disease (KD) as a marker for detecting, monitoring and predicting KD. Currently, the measure is not standardized. The variability of results obtained with commercially available procedures is important and can lead to misclassification of patients. Analytical standardization, started in 2007, is in progress. SRM 2925 primary reference material, SRM 3666 secondary reference material and liquid chromatography isotope dilution mass spectroscopy (LC-IDMS) reference measurement procedure are being validated by the National Institute of Standards and Technology (NIST). This report presents strategies and difficulties for developing this reference system.La mesure de la microalbuminurie a été introduite dans la nouvelle classification de la maladie rénale (MR) comme marqueur pour le dépistage, le suivi et la prédiction du risque. Actuellement la mesure est non standardisée.La variabilité des résultats obtenus avec différentes trousses commercialisées est importante et peut entraîner des erreurs de classification des patients. La standardisation analytique, débutée en 2007, progresse. Un matériau de référence primaire SRM 2925, un matériau de référence secondaire SRM 3666et une méthode de mesure de référence, la chromatographie liquide couplée à la spectrométrie de masse avec dilution isotopique (LC-IDMS), sont en cours de validation au National institute of standards and technology(NIST). Cet article présente les stratégies et les difficultés pour mettre au point ce système de référence

Harmonization and standardization of biofluid‐based biomarker measurements for AT(N) classification in Alzheimer's disease

Abstract Fluid biomarkers are currently measured in cerebrospinal fluid and blood for Alzheimer's disease diagnosis and are promising targets for drug development and for patients’ follow‐up in clinical trials. These biomarkers have been grouped in an unbiased research framework, the amyloid (Aβ), tau, and neurodegeneration (AT[N]) biomarker system to aid patients’ early diagnosis and stratification. Metrological approaches relying on mass spectrometry have been used for the development of reference materials and reference measurement procedures. Despite their excellent performances as clinical tools, fluid biomarkers often present an important between‐laboratory variation. Standardization efforts were carried out on the biomarkers currently included in the AT(N) classification system, involving the collaboration of national metrology institutes, clinicians, researchers, and in vitro diagnostic providers. This article provides an overview of current activities towards standardization. These reference methods and reference materials may be used for recalibration of immunoassays and the establishment of standardized cutoff values allowing a better stratification of Alzheimer's disease patients. Highlights The AT(N) biomarker system allows stratifying AD patients on the basis of biomarker profiles. Fluid biomarker measurements often present an important between‐laboratory variation preventing the establishment of standardized cutoff values. Overview on the standardization initiatives involving the fluid biomarkers currently included in the AT(N) framework

Candidate high-resolution mass spectrometry-based reference method for the quantification of procalcitonin in human serum using a characterized recombinant protein as a primary calibrator

International audienceProcalcitonin (PCT) is a widely used biomarker for rapid sepsis diagnosis and antibiotic stewardship. Variability of results in commercial assays has highlighted the need for standardization of PCT measurements. An antibody-free candidate reference measurement procedure (RMP) based on the isotope dilution mass spectrometry and protein calibration approach was developed and validated to quantify PCT in human serum. The method allows quantification of PCT from 0.25 to 13.74 μg/L (R > 0.998) with extension up to 132 μg/L after dilution of samples with PCT concentration above 13.74 μg/L. Intraday bias was between −3.3 and +5.7%, and interday bias was between −3.0 and −0.7%. Intraday precision was below 5.1%, and interday precision was below 4.0%. The candidate RMP was successfully applied to the absolute quantification of PCT in five frozen human serum pools. A recombinant PCT used as a primary calibrator was characterized by high-resolution mass spectrometry and amino acid analysis to establish traceability of the results to the SI units. This candidate RMP is fit to assign target values to secondary certified reference materials (CRMs) for further use in external quality assessment schemes to monitor the accuracy and comparability of the commercially available immunoassay results and to confirm the need for improving the harmonization of PCT assays. The candidate RMP will also be used to evaluate whether the correlation between the candidate RMP and immunoassays is sufficiently high. Overall, this candidate RMP will support reliable sepsis diagnosis and guide treatment decisions, patient monitoring, and outcomes

Impurity determination for hepcidin by liquid chromatography-high resolution and ion mobility mass spectrometry for the value assignment of candidate primary calibrators

International audienceIn metrology institutes, the state-of-the-art for purity analysis of peptides/proteins mainly addresses short and unfolded peptides. Important developments are anticipated for the characterization of nonlinear peptides or proteins. Hepcidin 1-25 is an interesting model system because this small protein contains four disulfide bridges with a particular connectivity that is difficult to reproduce and could induce a bias in quantification. Hepcidin 1-25 is involved in iron-related disorders and anemia, in an inflammatory context, and its clinical relevance in neurodegenerative disorders is under investigation. It is also an emerging biomarker. Recent inter-laboratory studies showed a need for standardization of hepcidin assay and the need to produce certified reference materials. This paper discusses two hepcidin standards from different synthesis pathways that have been characterized by high-resolution mass spectrometry and ion mobility mass spectrometry

La pratique clinique des psychologues en soins palliatifs, un référentiel francais

International audienceThe French Palliative Care Association (SFAP) has decided to take an in-depth look at the clinical practice of psychologists involved in palliative care. The nationwide study ran from 2012 to 2016. A previous study (Van Lander, 2012) analyzed the processes of 344 psychological follow-ups. Based on these data and the results of a questionnaire sent to all psychologists from French palliative care teams, a repository was produced. The editors were representative of field psychologists and experts in the field. The board of directors, scientific committee, the National College of Teachers for University Training in Palliative Care and the SFAP College of Psychologists were all involved and approved the 64 pages document. This document is straightforward, in the articulation between theory and practice. It is valid for the French context and constitutes a body of work that could be adapted for other European countries.La Société française de soins palliatifs (SFAP) a défini la pratique clinique des psychologues intervenant en soins palliatifs. L’étude nationale s’est déroulée de 2012 à 2016. Une précédente étude (Van Lander, 2012) analysait les processus de 344 suivis psychologiques. À partir de ces données et des résultats d’un questionnaire adressé à tous les psychologues des équipes françaises de soins palliatifs, un référentiel a été réalisé. Les rédacteurs étaient représentatifs des psychologues de terrain et experts dans le domaine. Le conseil d’administration, le comité scientifique, le Collège national des enseignants à la formation universitaire en soins palliatifs et le Collège des psychologues de la SFAP ont approuvé le document final de 64 pages. Ce document est simple, dans l’articulation entre théorie et pratique. Validé pour la France, il constitue une base de travail à adapter aux pays européen