Die vorliegende Arbeit quantifiziert die Korrelationen zwischen blutrheologischen Parametern und Erythrozyten-Indizes an einem repräsentativen und gut definierten Kollektiv gesunder Frauen im Vorfeld eines elektiven gynäkologischen Eingriffes. Gemäß der erhobenen Resultate besteht bei 286 gesunden Frauen mittleren Alters zwischen der Verformbarkeit, dem MCV und dem MCH der Erythrozyten eine statistisch signifikante Korrelation, die in Gegenwart hoher Scherkräfte an Ausprägung zunimmt. Gleichzeitig vermindert sich mit zunehmender MCHC die Erythrozytenverformbarkeit stetig und statistisch signifikant, wobei dieser Effekt am deutlichsten in Gegenwart hoher Scherkräfte, wie diese beispielsweise in den Kapillaren der Endstrombahn anzunehmen sind, nachweisbar ist. / The physiological association between blood rheological parameters and red blood cell indices was investigated in otherwise healthy unselected women prior to elective gynaecological surgery. In a total of 286 healthy women was the RBCdeformability statically significantly positive correlated with MCV and MCH but statically significantly inversely correlated with MCHC. The correlation between RBC Indices and RBC deformability was most remarkable during moderate shear force exposure.

Cancer is a major health issue that has been associated with over 80 million deaths worldwide in the last decade. Recently, significant improvements have been made in terms of treatment and diagnosis. However, despite these advancements there is still a demand for low-cost, high-accuracy, and easy-to-use technologies capable of classifying cells. Analysis of cell behavior in microfluidic deformability assays provides a label-free method of observing cell response to physical and chemical stimuli. This body of work shows advancements made toward reaching our goal of a robust and cost-effective biosensing device that allows for the identification of normal and cancer cells. These devices can also monitor cell responses to physical and chemical stimuli in the form of mechanical deformation and chemotherapeutic drugs, respectively. Our initial design was a microfluidic device that consisted of three channels with varying deformation and relaxation regions. Cell velocities from the deformations regions allowed us to distinguish between normal and cancer cells at the single-cell level. The next design used a singular deformation channel that was embedded with an array of electrodes in order to measure entry time, transit time and velocities as a single cell passes through the channel. These factors were found to reveal information about the biomechanical properties of single cells. Embedded electrodes were implemented in order to reduce post processing times of the data analysis and provide more insight into the bioelectrical information of cells. Finally, we report a microfluidic device with parallel deformation channels and a single electrode pair to improve throughput and automate data collection of deformability assays. This thesis demonstrates how microfluidic deformability assays, with and without embedded electrodes, show promising capabilities to classify different cells based on their biophysical traits which can be utilized as a valuable tool for testing responses to physical and chemical stimuli. / MS / Cancer is a worldwide health issue with approximately 1.7 million new cases each year in the United State alone. Although a great amount of research has been conducted in this field, the numerous uncertainties and heterogeneity among tumors, which is amplified by the large diversity between patients, has limit progress in both diagnostics and therapy. Traditionally, cancer studies have primarily focused on biological and chemical techniques. However, more recently, researchers have begun to leverage engineering techniques to acquire a new perspective on cancer to better understand the underlying biophysical attributes. Thus far, various engineering methodologies have produced meaningful results, but these techniques are costly and tend to be laborious. As a result, there is a need for low-cost, high-accuracy, and easy-to-use technologies to aid with cancer research, diagnostics, and treatment. An emerging field to alleviate these concerns is microfluidics, which is a science involving the flow of fluids in micro-scale channels. The field of microfluidics shows a great deal of promise for the development of clinically ready devices for analyzing cancer cells at both the population and single cell levels. Investigating the behavior of cancer cells at a single cell level can provide valuable information to help better understand the responsiveness of tumors to physical or chemical stimuli, such as chemotherapeutic drugs. This thesis reports multiple robust and cost-effective biomedical micro-devices that are used to analyze normal and cancerous cells. These devices consist of a microfluidic channel with sensors and are created using micro-fabrication techniques. The unique designs have enabled the evaluation of cells based on their mechanical and electrical properties. Specifically, the mechanical properties can be measured by forcing a cell into a microfluidic channel that is smaller than the diameter of the cell and recording its response to this physical stimulus. Electrical properties are measured simultaneously as the cells are probed for their mechanical properties. In general, the mechanical and electrical properties of cells can be altered when they undergo internal change (i.e. diseased cells) or experience external stimuli. Thus, these properties can be utilized as indicators of cancer progression and can be used to distinguish tumorigenic from non-tumorigenic cells. Data collection from these devices is automated, allowing for the rapid acquisition of mechanical and electrical properties of cells with minimal post-processing. Results from these devices have been promising in their ability to indicate significant differences among various normal and cancer populations based on their mechanical and electrical attributes.

A deformabilidade é a característica que permite ao eritrócito normal de 7 a 8 micrômetros (m) circular por capilares de até 3m de diâmetro. Esse fenômeno depende da geometria celular, da viscosidade interna e das propriedades visco-elásticas da membrana eritrocitária. Dentre as várias técnicas de estudo da deformabilidade eritrocitária (DE), como: a aspiração por micropipeta, a filtração e a reoscopia, destaca-se a ectacitometria. Essa técnica utiliza um viscosímetro de fluxo laminar onde as modificações de forma dos eritrócitos são monitoradas continuamente por um feixe de raio LASER, processadas por microcomputador e inseridas em gráfico para posterior análise. A ectacitometria fornece o Índice de Deformabilidade (ID), o qual proporciona a medida da eliptocitogênese dos eritrócitos quando submetidos a uma força denominada shear stress. A anemia ferropriva é uma patologia muito freqüente na prática médica. Apresenta anormalidades morfológicas expressivas como: microcitose, hipocromia, ovalócitos, eliptócitos e hemácias em alvo. Alterações de deformabilidade eritrocitária foram descritas em diversas situações como na esferocitose hereditária, eliptocitose hereditária e anemias hemolíticas auto-imune. Na anemia ferropriva os trabalhos de deformabilidade eritrocitária são controversos. O presente estudo avalia a DE, utilizando a técnica da ectacitometria, em 21 pacientes portadores de anemia ferropriva documentada, antes e depois do tratamento com sais de ferro. Embora o tratamento da anemia tenha sido eficaz (Hb antes- 8,52 g/dl e Hb depois -12,74 g/dl), alguns pacientes persistiram com morfologia eritrocitária alterada. Os resultados demonstram DE diminuída em pacientes portadores de anemia ferropriva, quando comparada ao grupo controle (p< 0,0007). A ausência de normalização e manutenção da diferença estatística após a terapêutica (p< 0,03), em baixos valores de shear stress, pode ser atribuida à manutenção das alterações morfológicas eritrocitárias. Não foi verificada correlação entre o grau da anemia e a redução da DE. A diminuição da DE apresenta maior correlação com a microcitose, sendo que a hipocromia parece não interferir de maneira importante. A concomitância dos dois fatores pode somar ou anular os seus efeitos sobre a DE. O presente estudo sugere que o fator responsável pela diminuição da DE na anemia ferropriva é a microcitose. Recentemente, relatos de anemia ferropriva associada a fenômenos trombóticos aumentaram o interesse no estudo da DE para melhor compreensão desses casos. / The deformability allows the 7 to 8 cm red cell to circulate through capillaries of 3 cm. This phenomenon depends on cellular geometry, internal viscosity and viscoelastic properties of the membrane. Among the various techniques of erytrocyte deformability (ED) analysis such as: micropipette aspiration, filtration and reoscopy, we chose ektacytometry. This technique uses a laminar flow viscometry, where red cell shape changes are continuously monitorated by LASER, processed by a computer and inserted in a graphic for further analysis. Ektacytometry measures the Deformability Index (DI), which shows the size of eliptocytogenesis of the eritrocyte under shear stress force. Iron deficiency anemia is a very frequent disease in medical practice. It presents expressive morphologic alterations such as microcytosis, hypocromy, ovalocytosis, eliptocytosis and target cells. Erytrocyte deformability has been described in a number of situations like hereditary spherocytosis, hereditary elitptocytosis and autoimmune hemolytic anemia. Concerning iron deficiency anemia, authors are controversial. The present study evaluates erytrocyte deformability, using ectacytometry in 21 patients carrying documented iron deficiency before and after therapeutics with iron components. Although the anemia treatment proved to be efficient (before Hb- 8,52 g/dl and after Hb- 12,74 g/dl), some patients persisted with erytrocyte alteration morfology. Results demonstrate diminished erytrocyte deformability in people with iron deficiency anemia, when compared with the control group (p< 0,0007). The absence of regularization and maintenance of statistical difference after treatment (p< 0,03) in low shear stress can be attributed to the persistence of red cell anomalies. There is no relation between the level of anemia and reduced ED. The erytrocyte deformability diminished is greatly related to microcitosys, even if hipocromy seems to not interefere importantly. The two factors altogether can either sum or nulify the effects over erytrocyte deformability. The present study suggests that the responsible factor for diminished erytrocyte deformability is microcitosys. Recently, iron deficiency anemia has been associated to thrombotic phenomenon has raised interest in the studying of erytrocyte deformability, in order to understand such cases.

A deformabilidade é a característica que permite ao eritrócito normal de 7 a 8 micrômetros (m) circular por capilares de até 3m de diâmetro. Esse fenômeno depende da geometria celular, da viscosidade interna e das propriedades visco-elásticas da membrana eritrocitária. Dentre as várias técnicas de estudo da deformabilidade eritrocitária (DE), como: a aspiração por micropipeta, a filtração e a reoscopia, destaca-se a ectacitometria. Essa técnica utiliza um viscosímetro de fluxo laminar onde as modificações de forma dos eritrócitos são monitoradas continuamente por um feixe de raio LASER, processadas por microcomputador e inseridas em gráfico para posterior análise. A ectacitometria fornece o Índice de Deformabilidade (ID), o qual proporciona a medida da eliptocitogênese dos eritrócitos quando submetidos a uma força denominada shear stress. A anemia ferropriva é uma patologia muito freqüente na prática médica. Apresenta anormalidades morfológicas expressivas como: microcitose, hipocromia, ovalócitos, eliptócitos e hemácias em alvo. Alterações de deformabilidade eritrocitária foram descritas em diversas situações como na esferocitose hereditária, eliptocitose hereditária e anemias hemolíticas auto-imune. Na anemia ferropriva os trabalhos de deformabilidade eritrocitária são controversos. O presente estudo avalia a DE, utilizando a técnica da ectacitometria, em 21 pacientes portadores de anemia ferropriva documentada, antes e depois do tratamento com sais de ferro. Embora o tratamento da anemia tenha sido eficaz (Hb antes- 8,52 g/dl e Hb depois -12,74 g/dl), alguns pacientes persistiram com morfologia eritrocitária alterada. Os resultados demonstram DE diminuída em pacientes portadores de anemia ferropriva, quando comparada ao grupo controle (p< 0,0007). A ausência de normalização e manutenção da diferença estatística após a terapêutica (p< 0,03), em baixos valores de shear stress, pode ser atribuida à manutenção das alterações morfológicas eritrocitárias. Não foi verificada correlação entre o grau da anemia e a redução da DE. A diminuição da DE apresenta maior correlação com a microcitose, sendo que a hipocromia parece não interferir de maneira importante. A concomitância dos dois fatores pode somar ou anular os seus efeitos sobre a DE. O presente estudo sugere que o fator responsável pela diminuição da DE na anemia ferropriva é a microcitose. Recentemente, relatos de anemia ferropriva associada a fenômenos trombóticos aumentaram o interesse no estudo da DE para melhor compreensão desses casos. / The deformability allows the 7 to 8 cm red cell to circulate through capillaries of 3 cm. This phenomenon depends on cellular geometry, internal viscosity and viscoelastic properties of the membrane. Among the various techniques of erytrocyte deformability (ED) analysis such as: micropipette aspiration, filtration and reoscopy, we chose ektacytometry. This technique uses a laminar flow viscometry, where red cell shape changes are continuously monitorated by LASER, processed by a computer and inserted in a graphic for further analysis. Ektacytometry measures the Deformability Index (DI), which shows the size of eliptocytogenesis of the eritrocyte under shear stress force. Iron deficiency anemia is a very frequent disease in medical practice. It presents expressive morphologic alterations such as microcytosis, hypocromy, ovalocytosis, eliptocytosis and target cells. Erytrocyte deformability has been described in a number of situations like hereditary spherocytosis, hereditary elitptocytosis and autoimmune hemolytic anemia. Concerning iron deficiency anemia, authors are controversial. The present study evaluates erytrocyte deformability, using ectacytometry in 21 patients carrying documented iron deficiency before and after therapeutics with iron components. Although the anemia treatment proved to be efficient (before Hb- 8,52 g/dl and after Hb- 12,74 g/dl), some patients persisted with erytrocyte alteration morfology. Results demonstrate diminished erytrocyte deformability in people with iron deficiency anemia, when compared with the control group (p< 0,0007). The absence of regularization and maintenance of statistical difference after treatment (p< 0,03) in low shear stress can be attributed to the persistence of red cell anomalies. There is no relation between the level of anemia and reduced ED. The erytrocyte deformability diminished is greatly related to microcitosys, even if hipocromy seems to not interefere importantly. The two factors altogether can either sum or nulify the effects over erytrocyte deformability. The present study suggests that the responsible factor for diminished erytrocyte deformability is microcitosys. Recently, iron deficiency anemia has been associated to thrombotic phenomenon has raised interest in the studying of erytrocyte deformability, in order to understand such cases.

Nous avons étudié les marqueurs hémorhéologiques, hématologiques et biochimiques chez des sujets drépanocytaires homozygotes SS (HbS/HbS) et hétérozygotes composites SC (HbS/HbC) dans deux cohortes, pédiatriques et adultes, de patients drépanocytaires, et ce, à travers 7 complications récurrentes de la drépanocytose : 2 appartenant au profil hémolytique (l’ulcère de jambes et la glomérulopathie) et 5 appartenant au phénotype visqueux/vaso-occlusif (l’hypertension artérielle, le syndrome thoracique aigu (STA), la crise vaso-occlusive (CVO), la rétinopathie et l’ostéonécrose). Nous avons montré que : 1) une viscosité sanguine et une déformabilité érythrocytaire élevées sont des facteurs de risques de CVO chez les enfants homozygotes ; 2) Une viscosité sanguine élevée est associée à une hypertension artérielle systémique relative chez des adultes SS ; 3) les enfants SC présente une fonction vasculaire mieux préservée que les enfants SS pour faire face à une augmentation de la viscosité sanguine ; 4) les patients adultes SS avec une ostéonécrose présentent une déformabilité érythrocytaire plus élevée que les patients sans ostéonécrose ; 5) une viscosité sanguine élevée est associée à la présence d’une rétinopathie chez les adultes SC mais pas chez les SS ; 6) les patients adultes SS présentant une glomérulopathie ont un taux d’hémolyse élevé, une déformabilité érythrocytaire réduite et des agrégats érythrocytaires très robustes ; 7) les patients adultes SS avec des ulcères de jambes récurrents ont un taux d’hémolyse accru et une déformabilité érythrocytaire réduite. De plus, nos travaux confirment que l’-thalassémie module les propriétés de déformabilité érythrocytaire, mais montrent pour la première fois qu’elle module aussi les propriétés d’agrégation érythrocytaire, et notamment la force des agrégats érythrocytaires. En conclusion, ces travaux permettent de préciser le rôle de la rhéologie sanguine dans un certain nombre de complications de la drépanocytose et d’enrichir le modèle préexistant divisant les complications de la drépanocytose selon 2 phénotypes : hémolytique versus visqueux/vaso-occlusif. Nous montrons pour la première fois que le phénotype hémolytique est caractérisé aussi par des anomalies de la rhéologie du globule rouge : rigidité accrue et agrégats érythrocytaire robustes. / Hemorheological, hemathological, and biochemical marquers of patients with sickle cell anemia (SS) and patients with sickle cell SC disease (SC) were studied in 2 cohorts: children and adults. We focused on 7 recurrent complications: 5 belonging to the viscosity/vaso-occlusion phenotype (systemic hypertension, acute chest syndrome (ACS), vaso-occlusive crisis (VOC), retinopathy and osteonecrosis) and 2 belonging to the hemolytic phenotype (leg ulcer and glomerulopathy). Our results show that 1) high viscosity is associated with increased risk for VOC in SS children; 2) blood viscosity is increased in SS adults with systemic relative hypertension; 3) SC children have preserved vascular function compared to SS children; 4) SS adults with osteonecrosis are characterized by higher red blood cell (RBC) deformability than SS adults without osteonecrosis; 5) high blood viscosity is associated with retinopathy in SC adults but not in SS adults; 6) SS adults affected by glomerulopathy have high hemolytic rate, low RBC deformability and increased RBC aggregates strenght; 7) SS adults with recurrent leg ulcers have high hemolytic rate and reduced RBC deformability. Moreover, our studies shows that alpha-thalassemia modulate RBC deformability and RBC aggregation properties. In conclusion, this work shows for the first time that the hemolytic phenotype is characterized by an abnormal RBC rheology which may play a role in several sickle cell complications.

Objective: This pilot study explores the influence of acute alcohol exposure on cell mechanical properties of steady-state and activated leukocytes conducted with real-time deformability cytometry. Methods: Nineteen healthy male volunteers were enrolled to investigate the effect of binge drinking on biophysical properties and cell counts of peripheral blood leukocytes. Each participant consumed an individualized amount of alcohol to achieve a blood alcohol concentration of 1.2 ‰ as a mean peak. In addition, we also incubated whole blood samples from healthy donors with various ethanol concentrations and performed stimulation experiments using lipopolysaccharide and CytoStim™ in the presence of ethanol. Results: Our findings indicate that the biophysical properties of steady-state leukocytes are not significantly affected by a single episode of binge drinking within the first two hours. However, we observed significant alterations in relative cell counts and a shift toward a memory T cell phenotype. Moreover, exposure to ethanol during stimulation appears to inhibit the cytoskeleton reorganization of monocytes, as evidenced by a hindered increase in cell deformability. Conclusion: Our observations indicate the promising potential of cell mechanical analysis in understanding the influence of ethanol on immune cell functions. Nevertheless, additional investigations in this field are warranted to validate biophysical properties as biomarkers or prognostic indicators for alcohol-related changes in the immune system.

Objective: The study aimed to investigate the effect of exercise on immune cell count and cell mechanical properties in people with multiple sclerosis (pwMS) on different disease-modifying treatments (DMT) vs. healthy controls (HCs). Methods: A cohort of 16 HCs and 45 pwMS, including patients with lymphopenia (alemtuzumab and fingolimod) as well as increased lymphocyte counts (natalizumab), was evaluated for exercise-mediated effects on immune cell counts and lymphocyte deformability. As exercise paradigms, climbing stairs at normal speed or as fast as possible and cycling were used, while blood samples were collected before, immediately, and 20 as well as 60 min post-exercise. Immune cell subtypes and lymphocyte deformability were analyzed using multicolor flow cytometry and real-time deformability cytometry. Results: An increase in lymphocytes and selected subsets was observed following exercise in HCs and all pwMS on different DMTs. Patients with lymphopenia exhibited an increase in absolute lymphocyte counts and immune cell subsets till just below or into the reference range. An increase above the upper limit of the reference range was detected in patients on natalizumab. Exercise-induced alterations were observable even in low and more pronounced in high-intensity physical activities. Lymphocyte deformability was found to be only mildly affected by the investigated exercise regimes. Conclusion: People with multiple sclerosis (PwMS) treated with alemtuzumab, fingolimod, and natalizumab respond to acute exercise with a comparable temporal pattern characterized by the increase of immune cell subsets as HCs. The magnitude of response is influenced by exercise intensity. Exercise-mediated effects should be considered when interpreting laboratory values in patients on immunomodulatory therapy. The impact of exercise on biophysical properties should be further elucidated.