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Physical Charaterization of Red Blood Cell Aggregation Daniel Amadeus Dominic Flormann

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Physical Charaterization of Red Blood Cell Aggregation Daniel Amadeus Dominic Flormann Physical charaterization of red blood cell aggregation Daniel Amadeus Dominic Flormann To cite this version: Daniel Amadeus Dominic Flormann. Physical charaterization of red blood cell aggregation. Biological Physics [physics.bio-ph]. Universität des Saarlandes, 2017. English. NNT : 2017GREAY002. tel- 01577838 HAL Id: tel-01577838 https://tel.archives-ouvertes.fr/tel-01577838 Submitted on 28 Aug 2017 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. THÈSE Pour obtenir le grade de DOCTEUR DE LA COMMUNAUTÉ UNIVERSITÉ GRENOBLE ALPES préparée dans le cadre d’une cotutelle entre la Communauté Université Grenoble Alpes et l’Universität des Saarlandes Spécialité : Physique pour les sciences du vivant Arrêté ministériel : 25 mai 2016 Présentée par Daniel Amadeus Dominic Flormann Thèse dirigée par M. Thomas Podgorski et M. Christian Wagner préparée au sein du Laboratoire Interdisciplinaire de Physique, Grenoble et de Experimentalphysik, Saarbrücken dans les Écoles doctorales de Physique de l’Université Grenoble Alpes et de Dekanat der Universität des Saarlandes La caractérisation physique de l’agrégation des globules rouges Thèse soutenue publiquement le 29 mars 2017 , devant le jury composé de : Monsieur Pelster Rolf Professeur, Saarbrücken, Président Monsieur Abdul Barakat Directeur de recherche, CNRS délégation Ile-de-France Sud, Rapporteur Monsieur Stephan Gekle Professeur, Université de Bayreuth, Rapporteur Monsieur Claude Verdier Directeur de recherche, CNRS délégation Alpes, Membre Physical characterization of red blood cell aggregation Dissertation im Rahmen eines Cotutelle-Verfahrens zur Erlangung des Grades des Doktors der Naturwissenschaftslehre der Naturwissenschaftlich-Technischen Fakultät der Universität des Saarlandes Thèse dans le cadre d’une cotutelle pour obtenir le grade de Docteur de l’Université Grenoble Spécialité: Physique pour les sciences du vivant et de Doktor der Naturwissenschaften der Universität des Saarlandes von/présentée par Daniel Amadeus Dominic Flormann Saarbrücken/Grenoble 2016 Date and place of defense: 29th of March 2017, Saarland University President of the jury: Prof. Dr. Rolf Pelster Rapporteur: Prof. Dr. Abdul Barakat Rapporteur: Jun.-Prof. Dr. Stephan Gekle Examinateur: Prof. Dr. Claude Verdier Examinateur: Dr. Thomas Podgorski Examinateur: Prof. Dr. Christian Wagner Eidesstattliche Versicherung Hiermit versichere ich an Eides statt, dass ich die vorliegende Arbeit selbstständig und ohne Benutzung anderer als der angegebenen Hilfsmittel angefertigt habe. Die aus anderen Quellen oder indirekt übernommenen Daten und Konzepte sind unter Angabe der Quelle gekennzeichnet. Die Arbeit wurde bisher weder im In- noch im Ausland in gleicher oder ähnlicher Form in einem Verfahren zur Erlangung eines akademischen Grades vorgelegt. Saarbrücken, 25.11.2016 Daniel Flormann I Abstract Abstract In this work, five aspects of the red blood cell aggregation induced by macromolecules are investigated. A rheological approach focused on the normalization of viscosity as a function of the macromolecular adsorption rates using a commercial rheometer is proposed. Derived from that approach, the yield stress of aggregating red blood cell suspensions is investigated. The sedimentation rates of the utilized biological system are then studied. Microscopical investigations, including measurements of the microscopical aggregation index, lead to the conclusion that the C-reactive pro- tein, a plasma protein, does not influence the aggregation behavior of red blood cells. Detailed microscopical studies on the morphology of the interaction zones of aggregated red blood cells show that these strongly depend on the macromolecular concentration in good agreement with numerical simulations that allow to derive an approximation of the interaction energies. The latter are also directly measured with single cell force spectroscopy using an atomic force microscope with the additional result that the viscosity of the surrounding medium can influence the results sig- nificantly. Finally, the physical origin of aggregation is discussed and supported by several additional measurements. This allows to combine two existing theories and explain the bell-shape of interaction energy versus macromolecular concentration curve in a new way. Résumé Ce travail a été réalisé autour de cinq aspects de l’agrégation des globules rouges (RBCs) sanguins induite par des macromolécules. Une approche rhéologique, ciblée sur la normalisation de la viscosité en fonction du taux d’adsorption des macro- molécules et mesurée par un rhéomètre commercial, est proposée. Par cette ap- proche, la contrainte seuil de suspensions de cellules sanguines agrégées est aussi éval- uée. De plus, les taux de sédimentation des solutions biologiques utilisées sont aussi mesurés. Nos données microscopiques, incluant des mesures d’indice d’agrégation microscopique, ont eu pour conclusion que la protéine C réactive, une protéine du plasma, n’a pas d’influence sur le phénomène d’agrégation des RBCs. Des mesures microscopiques détaillées de la morphologie des zones de contact des RBCs ont mon- trées que ces dernières dépendent fortement de la concentration de macromolécules, III Chapter 0 en accord avec des simulations numériques dont ont pu être extraites des valeurs d’énergie d’interaction. Ces dernières ont en outre pu être directement mesurées par microscopie à force atomique avec pour résultat supplémentaire que la vis- cosité du milieu peut influencer la mesure de manière significative. Enfin, l’origine physique de l’agrégation est discutée et confirmée par des mesures additionnelles. Ceci permet de concilier deux théories et permet d’expliquer la forme en cloche de l’énergie d’interaction en fonction de la concentration en macromolécules d’une nouvelle manière. Kurzzusammenfassung In dieser Arbeit werden fünf Aspekte der makromolekül-induzierten Aggregation roter Blutzellen untersucht. Ein rheologischer Ansatz unter Verwendung eines kom- merziellen Rheometers behinhaltet die Viskositätsnormierung als Funktion der Ad- sorptionsraten von Makromolekülen. Hiervon abgeleitet werden die Fließspannung von Suspensionen aggregierter roter Blutzellen untersucht. Außerdem werden die Sedimentationsraten des verwendeten biologischen Systems analysiert. Mikroskopis- che Untersuchungen, einschließlich des mikroskopischen Aggregationsindexes, führen zu dem Schluss, dass das C-reaktive Protein (ein Plasmaprotein) die Aggregation- seigenschaften roter Blutzellen nicht beeinflusst. Detaillierte mikroskopische Stu- dien der Morphologie von Interaktionszonen aggregierter roter Blutzellen zeigen eine starke Abhängigkeit von der molekularen Konzentration, was gut mit numerischen Simulationen, die eine Annäherung an die Interaktionsenergien zulassen, überein- stimmt. Letztere wurden direkt mit Einzelzellkraftmikroskopie unter Verwendung eines Atomkraftmikroskops gemessen, was zu dem Resultat führt, dass die Viskosität des umgebenden Mediums einen signifikanten Einfluss auf die Ergebnisse haben kann. Am Ende wird der physikalische Ursprung der Aggregation diskutiert und durch verschiedenen Messungen ergänzt. Dies erlaubt es, beide existierenden The- orien zu vereinen und die Glockenkurve der Interaktionsenergie als Funktion der Makromolekülkonzentration auf eine neue Weise zu erklären. IV Nomenclature AFM atomic force microscope BSA bovine serum albumin CCD charge-coupled device CMOS Complementary Metal Oxide Semiconductor CP cone-plate CR controlled rate CRP C-reactive protein CS controlled stress DC motor drag-cup motor Dex dextran DSLR digital single lens reflex camera EC motor electronically commutated synchronous motor EDTA ethylenediaminetetraaceticacid ESR erythrocyte sedimentation rate Fib Fibrinogen FITC fluorescein isothiocyanate HES hydroxyethylated starch HSA human serum albumin HyD high-sensitive hybrid detector IDT interdigital transducer IgG immunoglobulin G V Chapter 0 IgM immunoglobulin M kDa kilo-Dalton LSCM line scanning confocal microscopy MAI microscopical aggregation index MP megapixel NA numerical aperature PBS phosphate buffered saline PEG poly(ethylene glycol) PMT photomultiplier tube PSCM point scanning confocal microscopy RBC red blood cells SAW surface acoustic waves SCFS single cell force spectroscopy SD standard deviation SDCM spinning disk confocal microscopy SE standard error SMT seperate motor transducer TC Taylor-Couette TEM transmission electron microscopy UV/Vis ultraviolet/visible WBC white blood cells VI Contents Nomenclature V 1 Introduction 1 1.1 Introduction . 1 1.2 Literature overview . 8 2 Materials and methods 11 2.1 Blood, red blood cell aggregation introduction and theoretical back- round . 12 2.1.1 Blood and red blood cells . 12 2.1.2 Macromolecules . 16 2.1.3 Bridging and Depletion . 19 2.2 General techniques . 30 2.2.1 Confocal microscopy . 30 2.2.2 Rheology . 36 2.2.3 AFM . 44 2.2.4 UV/Vis Spectroscopy . 49 2.2.5 Surface acoustic waves (SAW) . 52 2.3 Measurement techniques and preparation protocols . 55 2.3.1 Sedimentation measurements . 55 2.3.2 Microscopical aggregation
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