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Dans le domaine du diagnostic in vitro, l'étape d'extraction de micro-organismes à partir d'un échantillon complexe est une étape clé pour permettre l'identification du pathogène responsable d'une infection. Pour les septicémies, cette étape d'extraction est généralement précédée d'une étape de culture, ce qui conduit à une obtention des résultats au bout de plusieurs jours. Un résultat plus rapide (typiquement inférieur à 24h) permettrait d'augmenter le taux de survie des patients, et aurait ainsi une forte valeur ajoutée pour le corps médical. Le but de ces travaux est donc de développer une nouvelle méthode d'extraction et de concentration de pathogènes directement à partir d'un échantillon sanguin, sans étape de culture. Une stratégie en deux modules microfluidiques associés en série est proposée : elle repose sur la modification de la conductivité et de l'osmolarité de l'échantillon dans un premier module, puis sur la capture des micro-organismes par diélectrophorèse dans un second module. L'étude du premier module a permis de déterminer l'impact de la conductivité et de l'osmolarité du milieu sur les propriétés diélectriques des cellules. Deux voies ont ainsi été abordées, afin de diriger les cellules du sang et les micro-organismes vers un milieu de conductivité et d'osmolarité contrôlées : la dilution, et l'utilisation de forces acoustiques. L'étude du deuxième module a ensuite permis de démontrer la possibilité de capturer et concentrer des micro-organismes à partir d'un échantillon hypotonique et faiblement conducteur dans un écoulement microfluidique par diélectrophorèse. L'architecture d'un microsystème dédié a été définie grâce à un modèle numérique, puis validé expérimentalement avec des échantillons sanguins et différents micro-organismes (E. coli, S. epidermidis et C. albicans). La capture générique des micro-organismes est démontrée, et un taux de capture de 97% a été obtenu pour la séparation de \EC, avec une vitesse moyenne de l'échantillon dans le microsystème de 100 à 200 µm.s-1. Enfin, des perspectives d'amélioration sont présentées pour permettre d'effectuer cette étape de séparation sur un gros volume d'échantillon (1 à 10mL) en quelques heures, afin de répondre aux exigences imposées par l'urgence des tests de diagnostic des septicémies. / Extraction of pathogens from a biological sample is a key step for efficient diagnostic tests of infectious diseases. For bloodstream infections, current diagnostic methods are usually based on bacterial growth and take several days to provide valuable information. An accelerated result would have a high medical value to adjust therapeutic strategies. The aim of this study is to design a new approach for separation and concentration of microorganisms directly from a blood sample, to avoid time-consuming growth stages. We report a method based on two different microsystems connected in series: it combines modification of conductivity and osmolarity of the sample with generic capture of microorganisms by dielectrophoresis. First we explore the impact of conductivity and osmolarity on the dielectric properties of blood cells and microorganisms. Dilution and acoustic forces are both analyzed to transfer blood cells and microorganisms to the optimized buffer. Then we demonstrate the feasibility of achieving the dielectrophoretic separation of microorganisms from blood cells in a low conductivity and low osmolarity medium inside a fluidic device. The structure of the device is optimized with numerical simulations and experiments performed on blood samples and various microorganisms (E. coli, S. epidermidis and C. albicans).The generic capture of microorganisms is validated, and we achieved a separation of 97% efficiency with E. coli, with an optimal inlet velocity around 100-200 µm.s-1. Finally, we propose an improved microsystem to perform the sample preparation step on a larger volume (1-10mL) in a few hours, in order to fit the medical need.

Les micro et nanoparticules magnétiques sont de plus en plus utilisées en biologie et en médecine, pour une large gamme d'applications. Plusieurs applications utilisent le piégeage et le guidage de ces billes sous l'effet d'un champ et d'un gradient de champ magnétique. Dans la plupart des applications, le champ magnétique est macroscopique, créé par un aimant ou un électro-aimant. L'intégration plus poussée est souvent envisagée, dans les articles scientifiques, par des microbobines ou par des éléments magnétiques doux. Ceux-ci doivent alors être polarisés par un champ externe (de nouveau, un électroaimant ou un aimant).Les micro-aimants mis au point à l'Institut Néel permettent d'obtenir les mêmes inductions que les meilleurs aimants du marché et, par conséquent, de par la réduction d'échelle, des gradients de champ intenses et donc des forces volumiques très conséquentes. Ils sont, de plus, favorables à l'autonomie et à la stabilité du système.Ce travail propose d'utiliser ces micro-aimants pour des applications en diagnostic In Vitro afin de tirer parti des forces volumiques importantes issues des micro-aimants et de la facilité d'utilisation de telles sources de champ magnétiques pour l'utilisateur.Ces premières constatations nous ont permis de mettre un place un test de type ELISA en une seule étape. Grâce à ces avantages, il a été possible d'utiliser des nanoparticules magnétiques à la place des classiques microparticules comme rapporté dans l'état de l'art. Ces nanoparticules, fonctionnalisables par des anticorps permettent entre autre d'augmenter le rapport surface sur volume phénomène très favorable à la sensibilité des tests de diagnostic In Vitro. De plus, les nanoparticules étant de petite taille, il est possible d'augmenter fortement leur concentration et de favoriser ainsi la capture de ces particules par les micro-aimants grâce à un mécanisme d'interaction fluide/particule et in fine la cinétique du test.Un autre avantage des micro-aimants permanents est la possibilité de contrôler le champ magnétique sur des distances micrométrique. Cela ouvre la voie à des tests de diagnostic sans lavage, simples et sensibles. Enfin, tous ces avantages ont été combinés à ceux de la microfluidique pour permettre l'émergence de test portables tout en restant efficaces. Pour cela l'autonomie intrinsèque aux micro-aimants permanents sera un avantage incontestable. / The range of applications for magnetic micro- and nanoparticles is constantly expanding, in particular in medicine and biology. A number of applications involve particle trapping and deviation under the effect of a magnetic field and field gradient. In most publications, the required magnetic fields are produced either using soft magnetic elements polarized by an external magnetic field, electromagnets or bulk permanent magnets.Micromagnets produce high fields and favor autonomy and stability while downscaling leads to an increase of field gradients and consequently increase strongly the forces.Micromagnets developped at the Neel Institute produce magnetic induction as good as the best macro-magnets. Therefore, thanks to scale reduction laws, high field gradients and therefore intense forces can be obtained. Moreover, these magnets can easily be integrated in micro systems such as BioMEMS.The purpose of this work is to use these micromagnets to develop in vitro immunoassays.. An innovative system based on superparamagnetic nanoparticles attraction by micromagnets was developed in order to perform a “one step” ELISA.Nanoparticles can be functionalized with antibodies, increasing the surface/volume ratio, and therefore the test sensitivity. Thanks to their small size, the nanoparticles concentration can be increased, and a fluid/particles interaction optimizes their capture by the micromagnets. This phenomena is favorable to immunoassay's kinetics.A micrometric control of the magnetic field is possible thanks to micromagnets: this allows to design simple and sensitive immunoassays that need no washing steps. Finally, these properties combined to microfluidics is used to design of point of care and sensitive immunoassays.

La dématérialisation de l’accès aux tests génétiques apparaît comme un outil concourant à satisfaire l’ensemble des composantes du droit à la protection de la santé. En effet, en la matière, la spécialisation de la médecine induit une limitation des personnes habilitées à prescrire des tests génétiques. Aussi, la recherche de la meilleure sécurité sanitaire possible pour la réalisation des tests génétiques induit des problématiques d’égal accès aux laboratoires de biologie médicale autorisés à cet effet, mais auxquelles la dématérialisation peut apporter des réponses. Aussi, elle n’apparaît pas être antinomique de l’autonomie des patients, bien qu’elle présente des faiblesses. / The dematerialization of access to genetic testing appears to be a tool to satisfy all the aspects of the right to health protection. Indeed, in this field, the specialization of medicine induces a limitation of the numbers of persons entitled to prescribe genetic tests. The quest for the best quality and health security for the realization of the genetic tests induces problems of equal access to the laboratories of medical biology authorized for this purpose, but to which dematerialization can give answers. Also, dematerialization does not appear to be antinomic of patient autonomy, although it presents weaknesses.

The newly introduced regulation (EU) 2017/746 aims to make In Vitro Diagnostic Medical Devices (IVDMD), which include companion diagnostic tests (CDx), a widespread method of authorising medicinal products in the European market. However, European SmPCs (Summary of Product Characteristics) currently do not explicitly refer to the term or classify tests associated with medicinal products as CDx. This paper is the first to examine and classify tests for medicinal products currently authorised in Sweden as being potential CDx, under the definitions of the new regulatory paradigm. The aim is to serve as the foundation for future research. 141 medicinal products with potential associated CDx are identified in the database of the Swedish Medical Products Agency (MPA). These products are then classified under the major ATC (Anatomical Therapeutic Chemical) therapeutic areas to search for commonalities and patterns in their usage and are later examined in conjunction with the techniques they use. The results reveal that a majority are concentrated in the Antineoplastic and immunomodulating agents and Antiinfectives for systemic use therapeutic areas. The methods used by these tests reveal diversity among the test technique usage, including instances where multiple techniques comprise a single CDx product, which may focus on detecting several biomarkers.

Du coeur artificiel au pansement en passant par les prothèses, lentilles correctrices, fauteuils roulants ou appareils de radiologie, la notion de dispositif médical recouvre un vaste ensemble de produits ayant en commun leur finalité médicale et leur action qui n’est pas obtenue par des moyens pharmacologiques ou immunologiques ni par métabolisme. Les dispositifs médicaux ont été règlementés dans les années 1990 par des directives fondées sur les principes de la nouvelle approche (définition d’exigences essentielles et renvoi à l’harmonisation technique, place importante laissée aux acteurs professionnels et rôle limité conféré aux autorités publiques, évaluation de la conformité des produits par des organismes notifiés et absence d’autorisation de mise sur le marché). Malgré les avantages liés à leur souplesse, ces directives n’ont qu’imparfaitement réussi à garantir la sécurité des dispositifs médicaux, ainsi que cela a été rappelé dans le contexte de l’affaire des prothèses PIP : évaluation clinique, information et traçabilité des produits insuffisantes, contrôle par et sur les organismes notifiés limité, faible coordination entre les autorités, etc. Pour renforcer la sécurité des dispositifs médicaux européens, plutôt qu’une transposition du cadre juridique des médicaments ou des dispositifs médicaux américains, un cadre juridique sur mesure, adapté à leur niveau variable de risque, est à envisager. C’est ce que proposent les règlements qui seront prochainement adoptés. / From artificial heart to bandage through implants, corrective lenses, wheelchairs or radiology devices, the concept of medical device covers a wide range of products having in common their medical purpose and their action which is not obtained by pharmacological, immunological or metabolic means. Medical devices were regulated in the 1990s by directives based on the principles of the new approach (definition of essential requirements and reference to technical harmonization, important role for professional actors and limited role for public authorities, evaluation of the conformity of products by notified bodies and absence of marketing authorization). Despite the advantages of their flexibility, these directives have only partially succeeded in guaranteeing the safety of medical devices, as underlined in the circumstances of PIP implants scandal: limited clinical evaluation, information and traceability of products, limited control by and on notified bodies, lack of coordination between authorities, etc. To strengthen the safety of European medical devices, rather than transposing the legal framework of medicinal products or American medical devices, a custom-made legal framework, adapted to their varying level of risk, is to be considered. This is what the upcoming regulations put forward.

During the last decades, newly developed medical devices often came along with unappropriate designs, increasing the likelihood of misuse through the operator. Part of the root cause was that no sufficient measures were applied to assimilate user needs. Consequently, usability engineering approaches are now stronger emphasized to ensure that new devices are not only safe to use but are also designed for users’ needs. Besides, testing processes in clinical microbiology laboratories are currently reshaped due to new generations of rapid testing methods. Hence, it is particularly important to apply usability engineering frameworks during the development phase to make sure devices address users’ needs and also fit into the new work- and communication flows. Based on that, this research project applies a usability engineering approach to the design process of a new rapid antibiotic susceptibility testing system of Astrego Diagnostic AB that is supposed to be used in clinical microbiology laboratories in the near future. The research questions focus on how this device can be designed to enable integration into clinical laboratories. -       How can a rapid AST testing system be integrated into the workflow of clinical microbiology laboratories? -       What are the remaining uncertainties for integrating a rapid AST system into the workflow of a clinical microbiology laboratory on the example of Astrego’s AST system? Several methods were used to address these questions, which include literature research, a competitive audit, subject matter interviews and semi-structured interviews, and observations of targeted users. The findings show that a rapid antibiotic susceptibility testing system may be used in several different ways, which also impacts its design. Process-wise, it could be used after Gram staining and bacterial identification has been conducted and, more realistically, simultaneously bacterial identification to pave the way for additional time savings further. However, uncertainties remain regarding the design of the new testing system. Depending on the number of devices that targeted laboratories need to implement to accommodate their testing volume, it makes sense to design a built-in user interface or an external one that can be accessed through a tablet or desktop. Thus, it is uncertain to what extent manual input of bacteria ID is relevant as the dRAST system fully enables manual input of Gram type and bacteria IDs while it might also be possible to avoid manual interaction by receiving this information through software interfaces.

Grâce à la spectrométrie de masse de type MALDI-TOF, l'identification des bactéries est maintenant possible en quelques minutes. Mais le taux de mortalité des patients augmente lorsqu'une antibiothérapie inappropriée est utilisée et les instruments MALDI-TOF ne sont pas capables d'analyser rapidement et exhaustivement la résistance bactérienne. Actuellement, 6 à 24 heures sont nécessaires pour déterminer le phénotype de résistance. En couplant une chromatographie liquide et un spectromètre de masse à ionisation électrospray (LC-MS/MS), nous avons identifié les marqueurs de résistance en 1 à 2 heures. En 30 min, nous avons pu détecter les mécanismes de résistance aux β-lactamines, aux glycopeptides, à la méthicilline et aux fluoroquinolones, à l'aide de méthodes de type "Suivi de Réactions Sélectionnées", ou "Selected Reaction Monitoring" (SRM). Au cours de la même analyse multiplexée, des dizaines de protéines peuvent être détectées de façon hautement spécifique et sensible. Comme l'illustre l'étude de la résistance multifactorielle chez Acinetobacter baumannii, cette approche permet en outre une analyse quantitative d'un grand intérêt pour certains mécanismes de résistance. Cependant, malgré ces perspectives attrayantes, la LC-MS/MS reste, aujourd'hui, loin d'une possible implantation en routine dans les laboratoires de microbiologie. Les instruments sont trop coûteux et la technologie trop complexe pour un usage pour du diagnostic in vitro. La spectrométrie de masse pourrait déjà avantageusement compléter les technologies actuelles de biologie moléculaire. Aujourd'hui, le séquençage de nouvelle génération est la méthode de référence pour la caractérisation moléculaire des bactéries. Mais, comme démontré dans ce travail, l'annotation des gènes est perfectible. Pour quelques dizaines d'euros et quelques heures d'analyse, les peptides identifies par spectrométrie de masse facilitent l'assemblage des séquences (« scaffolds ») et la détection des gènes. De surcroît, la spectrométrie de masse permet une quantification précise des protéines. Elle apporte ainsi une nouvelle dimension analytique et une vision moléculaire plus proche du phénotype. En conclusion, la spectrométrie de masse LC-MS/MS peut être une technologie complémentaire attractive, voir une future alternative, à la biologie moléculaire pour la caractérisation des bactéries / Thanks to MALDI-TOF mass spectrometry, identification of isolated bacteria is now possible within a few minutes. But doctors also need to rapidly know the phenotype of resistance of the bacteria. Indeed, the patient mortality rate increases when the antibiotherapy is not appropriate. However, MALDI-TOF instruments are not able to analyze antibacterial resistance rapidly and comprehensively.Today, 6 to 24 hours are nedded for antibiotic susceptibility testing. When combining a liquid chromatography and a mass spectrometer with electrospray ionization (LC-MSMS), the detection of resistance biomarkers was possible within 1 to 2 hours. Using a Selected Reaction Monitoring (SRM) method, resistance mechanisms to beta-lactams, methicillin, glycopeptides and fluoroquinolones were detected in strains within 30 minutes. Tens of resistance determinants can be analyzed in a single multiplexed assay, with high specificity and sensitivity. Illustrated by the study of multifactorial resistance in Acinetobacter baumannii, the technology allows furthermore a quantitative analysis, which is of great value for some resistance mechanisms. Similarly, we identified virulent strains of enterohemorrhagic Escherichia coli by targeting toxins and serotype biomakers in the same assay. Mass spectrometry offered deeper bacterial characterization than conventional serotyping using polyclonal antibodies. However, despite all these favorable prospects, LC-MS/MS remains today far from reaching a routine use in microbiological hospital laboratories. Instruments are too expensive and the technology is too cumbersome for a daily in vitro diagnostic use. Waiting for a more suitable use, mass spectrometry could yet advantageously complement current molecular technologies. Today, the gold standard to study bacteria at molecular level is next generation sequencing. However, as demonstrated during this work, gene annotation remains imperfect. For tens of euros and few hours of analysis, peptides identified by mass spectrometry analysis of a bacteria might improve scaffold assembly and gene detection. Moreover, mass spectrometry gives an accurate protein quantitation and brings a new analytical dimension, potentially closer to the phenotype than molecular techniques. In conclusion, LC-MS/MS mass spectrometry could be an attractive complementary, or alternative technology in a near future, to conventional molecular biology techniques for deep characterization of bacteria

Since 1994, the Public Procurement Act has undergone changes aimed to improve the procurement process and promoting innovation. In order to promote innovation there are a number of government agencies that offer financial assistance, especially to small business with innovative ideas. Despite these efforts, newly established small businesses have difficulties entering the market. The study investigates whether changes have been made in the public procurement of medical devices with regard to price and quality prioritization. The priority criteria is presented by the contracting authority as percentage in the allocation criteria. The study is limited to in vitro diagnostic products contracting in Swedish healthcare between the years 2009 and 2015. All tenders, companies that apply for participation in procurement, were analyzed based on sales, number of employees and year of establishment with the purpose of evaluating differences between winning tenders and other tenders. The result shows that prioritization has been increasingly on price rather than the quality. Regarding the winning tenders the study shows that larger, more established companies dominates the market. Noteworthy is that this dominance has increased over theses last years. The increase may be due to a spiral effect that is created when winning tenders get an increased economic power.

Metabolomics is the methodical scientific study of biochemical processes associated with the metabolome—which comprises the entire collection of metabolites in any biological entity. Metabolome changes occur as a result of modifications in the genome and proteome, and are, therefore, directly related to cellular phenotype. Thus, metabolomic analysis is capable of providing a snapshot of cellular physiology. Untargeted metabolomics is an impartial, all-inclusive approach for detecting as many metabolites as possible without a priori knowledge of their identity. Hence, it is a valuable exploratory tool capable of providing extensive chemical information for discovery and hypothesis-generation regarding biochemical processes. A history of metabolomics and advances in the field corresponding to improved analytical technologies are described in Chapter 1 of this dissertation. Additionally, Chapter 1 introduces the analytical workflows involved in untargeted metabolomics research to provide a foundation for Chapters 2 – 5. Part I of this dissertation which encompasses Chapters 2 – 3 describes the utilization of mass spectrometry (MS)-based untargeted metabolomic analysis to acquire new insight into cancer detection. There is a knowledge deficit regarding the biochemical processes of the origin and proliferative molecular mechanisms of many types of cancer which has also led to a shortage of sensitive and specific biomarkers. Chapter 2 describes the development of an in vitro diagnostic multivariate index assay (IVDMIA) for prostate cancer (PCa) prediction based on ultra performance liquid chromatography-mass spectrometry (UPLC-MS) metabolic profiling of blood serum samples from 64 PCa patients and 50 healthy individuals. A panel of 40 metabolic spectral features was found to be differential with 92.1% sensitivity, 94.3% specificity, and 93.0% accuracy. The performance of the IVDMIA was higher than the prevalent prostate-specific antigen blood test, thus, highlighting that a combination of multiple discriminant features yields higher predictive power for PCa detection than the univariate analysis of a single marker. Chapter 3 describes two approaches that were taken to investigate metabolic patterns for early detection of ovarian cancer (OC). First, Dicer-Pten double knockout (DKO) mice that phenocopy many of the features of metastatic high-grade serous carcinoma (HGSC) observed in women were studied. Using UPLC-MS, serum samples from 14 early-stage tumor DKO mice and 11 controls were analyzed. Iterative multivariate classification selected 18 metabolites that, when considered as a panel, yielded 100% accuracy, sensitivity, and specificity for early-stage HGSC detection. In the second approach, serum metabolic phenotypes of an early-stage OC pilot patient cohort were characterized. Serum samples were collected from 24 early-stage OC patients and 40 healthy women, and subsequently analyzed using UPLC-MS. Multivariate statistical analysis employing support vector machine learning methods and recursive feature elimination selected a panel of metabolites that differentiated between age-matched samples with 100% cross-validated accuracy, sensitivity, and specificity. This small pilot study demonstrated that metabolic phenotypes may be useful for detecting early-stage OC and, thus, supports conducting larger, more comprehensive studies. Many challenges exist in the field of untargeted metabolomics. Part II of this dissertation which encompasses Chapters 4 – 5 focuses on two specific challenges. While metabolomic data may be used to generate hypothesis concerning biological processes, determining causal relationships within metabolic networks with only metabolomic data is impractical. Proteins play major roles in these networks; therefore, pairing metabolomic information with that acquired from proteomics gives a more comprehensive snapshot of perturbations to metabolic pathways. Chapter 4 describes the integration of MS- and NMR-based metabolomics with proteomics analyses to investigate the role of chemically mediated ecological interactions between Karenia brevis and two diatom competitors, Asterionellopsis glacialis and Thalassiosira pseudonana. This integrated systems biology approach showed that K. brevis allelopathy distinctively perturbed the metabolisms of these two competitors. A. glacialis had a more robust metabolic response to K. brevis allelopathy which may be a result of its repeated exposure to K. brevis blooms in the Gulf of Mexico. However, K. brevis allelopathy disrupted energy metabolism and obstructed cellular protection mechanisms including altering cell membrane components, inhibiting osmoregulation, and increasing oxidative stress in T. pseudonana. This work represents the first instance of metabolites and proteins measured simultaneously to understand the effects of allelopathy or in fact any form of competition. Chromatography is traditionally coupled to MS for untargeted metabolomics studies. While coupling chromatography to MS greatly enhances metabolome analysis due to the orthogonality of the techniques, the lengthy analysis times pose challenges for large metabolomics studies. Consequently, there is still a need for developing higher throughput MS approaches. A rapid metabolic fingerprinting method that utilizes a new transmission mode direct analysis in real time (TM-DART) ambient sampling technique is presented in Chapter 5. The optimization of TM-DART parameters directly affecting metabolite desorption and ionization, such as sample position and ionizing gas desorption temperature, was critical in achieving high sensitivity and detecting a broad mass range of metabolites. In terms of reproducibility, TM-DART compared favorably with traditional probe mode DART analysis, with coefficients of variation as low as 16%. TM-DART MS proved to be a powerful analytical technique for rapid metabolome analysis of human blood sera and was adapted for exhaled breath condensate (EBC) analysis. To determine the feasibility of utilizing TM-DART for metabolomics investigations, TM-DART was interfaced with traveling wave ion mobility spectrometry (TWIMS) time-of-flight (TOF) MS for the analysis of EBC samples from cystic fibrosis patients and healthy controls. TM-DART-TWIMS-TOF MS was able to successfully detect cystic fibrosis in this small sample cohort, thereby, demonstrating it can be employed for probing metabolome changes. Finally, in Chapter 6, a perspective on the presented work is provided along with goals on which future studies may focus.