No description available.

Microhemodynamics is the study of blood flow in small vessels, usually on the order of 50 to 100 µm. The in vitro study of blood flow in small channels is analogous to the in vivo study of the microcirculation. At this scale the Reynolds and Womersly numbers are significantly less than 1 and the viscous stress and pressure gradient are the main determinant of flow. Blood is a non-homogeneous, non-Newtonian fluid and this complex composition and behavior has a greater impact at the microscale. A key parameter is the shear stress at the wall, which is involved in many processes such as platelet activation, gas exchange, embryogenesis and angiogenesis. In order to measure the shear rate in these blood flows the velocity profile must be measured. The measured profile can be characterized by the maximum velocity, the flow rate, the shear rate at the wall, or a shape parameter reflecting the bluntness of the velocity profile. The technique of micro-particle image velocimetry (µPIV) was investigated to measure the velocity profiles of blood microflows. The material of the channel, the type of tracer particles, the camera used, and the choice in data processing were all validated to improve the overall accuracy of µPIV as a blood microflow measurement method. The knowledge gained through these experiments is of immediate interest to applications such as the design of lab-on-a-chip components for blood analysis, analysis of blood flow behavior, understanding the shear stress on blood in the microcirculation and blood substitute analysis. Polymer channels were fabricated from polydimethylsiloxane (PDMS) by soft lithography in a clean room. PDMS was chosen for ease of fabrication and biocompatibility. The contacting properties of saline, water, and blood with various polymer channel materials was measured. As PDMS is naturally hydrophilic, surface treatment options were explored. Oxygenated plasma treatment was found to be less beneficial for blood than for water. The choice of camera and tracer particles were validated. Generally, for in vivo studies, red blood cells (RBCs) are used as tracer particles for the µPIV method, while for in vitro studies, artificial fluorescent micro particles are added to the blood. It is demonstrated here that the use of RBCs as tracer particles creates a large depth of correlation (DOC), which can approach the size of vessel itself and decreases the accuracy of the method. Next, the accuracy of each method is compared directly. Pulsed images used in conjunction with fluorescing tracer particles are shown to give results closest to theoretical approximations. The effect of the various post-processing methods currently available were compared for accuracy and computation time. It was shown that changing the amount of overlap in the post-processing parameters affects the results by nearly 10%. Using the greatest amount of correlation window overlap with elongated windows aligned with the flow was shown to give the best results when coupled with a image pre-processing method previously published for microflows of water. Finally the developed method was applied to a relevant biomedical engineering problem: the evaluation of blood substitutes and blood viscosity modifiers. Alginate is a frequently used viscosity modifier which has many uses in industry, including biomedical applications. Here the effect of alginate on the blood rheology, i.e., the shape of the velocity profile and the maximum velocity of blood flow in microchannels, was investigated. Alginate was found to blunt the shape of the velocity profile while also decreasing the shear rate at the wall. Overall, the accuracy of µPIV measurements of blood flows has been improved by this thesis. The work presented here has extended the known methods and accuracy issues of blood flow measurements in µPIV, improved the understanding of the blood velocity profile behavior, and applied that knowledge and methods to interesting, relevant problems in biomedical and biofluids engineering.

Στην παρούσα διπλωματική εργασία παρουσιάζεται μία μέθοδος αυτόματης αναγνώρισης των γεωμετρικών χαρακτηριστικών ερυθρών αιμοσφαιρίων από προσομοιωμένες εικόνες σκεδασμένου φωτός. Αρχικά παρουσιάζεται το ευθύ πρόβλημα σκέδασης ηλεκτρομαγνητικής ακτινοβολίας από ερυθρό αιμοσφαίριο, στη συνέχεια περιγράφονται οι τεχνικές με τις οποίες εκτελέστηκαν τα πειράματα για την επίλυση του αντίστροφου προβλήματος σκέδασης στο περιβάλλον προσομοίωσης του Matlab και τέλος προτείνεται μία μέθοδος έτσι ώστε η αναγνώριση να είναι δυνατή αυτόματα και σε πραγματικό χρόνο. / Geometric features recognition of red blood cells through scattered light pictures.

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 macromolécules et mesurée par un rhéomètre commercial, est proposée. Par cette approche, la contrainte seuil de suspensions de cellules sanguines agrégées est aussi évaluée. 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 montrées que ces dernières dépendent fortement de la concentration de macromolécules, 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 viscosité 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. / 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 protein, 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 significantly. Finally, the physical origin of aggregation is discussed and supported by several additional measurements. This allow to combine two existing theories and explain the bell-shape of interaction energy versus macromolecular concentration curve in a new way.

COSTA, Erivelton Façanha da. Caracterização de Células Vermelhas por Microscopia de Força Atômica. 2006. 146 f. Dissertação (Mestrado em Física) - Programa de Pós-Graduação em Física, Departamento de Física, Centro de Ciências, Universidade Federal do Ceará, Fortaleza, 2006. / Submitted by Edvander Pires (edvanderpires@gmail.com) on 2015-05-05T20:59:55Z No. of bitstreams: 1 2006_dis_efcosta.pdf: 10007653 bytes, checksum: e0be816e57ec642d42d294c9688b5c9f (MD5) / Approved for entry into archive by Edvander Pires(edvanderpires@gmail.com) on 2015-05-07T16:55:52Z (GMT) No. of bitstreams: 1 2006_dis_efcosta.pdf: 10007653 bytes, checksum: e0be816e57ec642d42d294c9688b5c9f (MD5) / Made available in DSpace on 2015-05-07T16:55:53Z (GMT). No. of bitstreams: 1 2006_dis_efcosta.pdf: 10007653 bytes, checksum: e0be816e57ec642d42d294c9688b5c9f (MD5) Previous issue date: 2006 / The optical microscopy is the most employed technique used for visualize red blood cells (RBCs). But, due to its resolution limit, it is necessary to use other complementary techniques to study the cells, such as: the scanning and transmission electron microscopy, and the scanning probing microscopy. The Atomic Force Microscopy (AFM) is included in the last group. This work refers to the possibilities of using AFM in life science, focusing on the erythrocytes characterization. Five experiments involving red blood cells and AFM were carried out: AB+ and O+ blood types differentiation; RBCs membrane study of donors and patients with MDS (Myelodysplastic Syndrome); suitable preparation of red blood cells for AFM analysis; volume study of erythrocytes; and finally aging process observation of RBC in air. The first experiment determined the cell membrane roughness for AB+ and O+ groups, which were different. For the second one, depressions were found on the cell surface of both MSD patients and healthy people. These "holes" were deeper in the first group. The third experiment showed the importance of sample (RBCs) preparation for each AFM specific analysis. The fourth experimental procedure showed the AFM technique capability for providing volume information, which was also used in the last experiment to monitor the aging process of RBCs in air. / A maneira mais difundida na observação de células sanguineas (hemácias) é aquela que utiliza microscopia ótica convencional. Devido ao limite de resolução dos instrumentos óticos, novas técnicas de microscopia colocam-se como alternativas para o estudo de células, tais como: a microscopia eletrônica (de varredura e transmissão) e as técnicas de varredura por sonda. Inclui-se neste último grupo a microscopia de força atômica (AFM). Este trabalho discute as possibilidades de uso da Microscopia de Força Atômica ($emph{Atomic Force Microscopy}$ - AFM) em ciências da vida, para ser mais específico, na caracterização de eritrócitos. Cinco experimentos envolvendo hemácias e AFM estão aqui descritos: diferenciação entre os grupos sanguíneos AB+ e O+; análise do perfil da membrana eritrocitária de indivíduos sadios e portadores de SMD; preparação de células vermelhas para análise em microscopia de força atômica; análise volumétrica de células vermelhas; e monitoramento do envelhecimento de um eritrócito ao ar usando o AFM. No primeiro experimento, a rugosidade das membranas celulares dos grupos AB+ e O+ mostraram-se diferentes. Já no segundo experimento, depressões foram encontradas sobre a membrana de pacientes com SMD e indivíduos sadios, contudo, aparentemente mais profundas no primeiro grupo. O terceiro estudo trouxe à tona a importância da preparação adequada dos eritrócitos para medidas específicas em AFM. A quarta experiência comprovou a capacidade da técnica AFM de fornecer informação de volume, o que também foi utilizado no último experimento para monitorar o envelhecimento de uma hemácia ao ar.

Microhemodynamics is the study of blood flow in small vessels, usually on the order of 50 to 100 µm. The in vitro study of blood flow in small channels is analogous to the in vivo study of the microcirculation. At this scale the Reynolds and Womersly numbers are significantly less than 1 and the viscous stress and pressure gradient are the main determinant of flow. Blood is a non-homogeneous, non-Newtonian fluid and this complex composition and behavior has a greater impact at the microscale. A key parameter is the shear stress at the wall, which is involved in many processes such as platelet activation, gas exchange, embryogenesis and angiogenesis. In order to measure the shear rate in these blood flows the velocity profile must be measured. The measured profile can be characterized by the maximum velocity, the flow rate, the shear rate at the wall, or a shape parameter reflecting the bluntness of the velocity profile. The technique of micro-particle image velocimetry (µPIV) was investigated to measure the velocity profiles of blood microflows. The material of the channel, the type of tracer particles, the camera used, and the choice in data processing were all validated to improve the overall accuracy of µPIV as a blood microflow measurement method. The knowledge gained through these experiments is of immediate interest to applications such as the design of lab-on-a-chip components for blood analysis, analysis of blood flow behavior, understanding the shear stress on blood in the microcirculation and blood substitute analysis. Polymer channels were fabricated from polydimethylsiloxane (PDMS) by soft lithography in a clean room. PDMS was chosen for ease of fabrication and biocompatibility. The contacting properties of saline, water, and blood with various polymer channel materials was measured. As PDMS is naturally hydrophilic, surface treatment options were explored. Oxygenated plasma treatment was found to be less beneficial for blood than for water. The choice of camera and tracer particles were validated. Generally, for in vivo studies, red blood cells (RBCs) are used as tracer particles for the µPIV method, while for in vitro studies, artificial fluorescent micro particles are added to the blood. It is demonstrated here that the use of RBCs as tracer particles creates a large depth of correlation (DOC), which can approach the size of vessel itself and decreases the accuracy of the method. Next, the accuracy of each method is compared directly. Pulsed images used in conjunction with fluorescing tracer particles are shown to give results closest to theoretical approximations. The effect of the various post-processing methods currently available were compared for accuracy and computation time. It was shown that changing the amount of overlap in the post-processing parameters affects the results by nearly 10%. Using the greatest amount of correlation window overlap with elongated windows aligned with the flow was shown to give the best results when coupled with a image pre-processing method previously published for microflows of water. Finally the developed method was applied to a relevant biomedical engineering problem: the evaluation of blood substitutes and blood viscosity modifiers. Alginate is a frequently used viscosity modifier which has many uses in industry, including biomedical applications. Here the effect of alginate on the blood rheology, i.e., the shape of the velocity profile and the maximum velocity of blood flow in microchannels, was investigated. Alginate was found to blunt the shape of the velocity profile while also decreasing the shear rate at the wall. Overall, the accuracy of µPIV measurements of blood flows has been improved by this thesis. The work presented here has extended the known methods and accuracy issues of blood flow measurements in µPIV, improved the understanding of the blood velocity profile behavior, and applied that knowledge and methods to interesting, relevant problems in biomedical and biofluids engineering.

Cryopreservation is a method used to preserve the quality of various cell types over long periods of time (up to several years). Using this preservation method can vastly improve cellular therapies and regenerative medicine by allowing the creation of biobanks containing high-quality cell products. For example, biobanks of red blood cells (RBCs) would be beneficial for cellular therapies such as RBC transfusions, which are used to treat patients suffering from hemorrhages, anemias, and to replace blood loss after traumatic/surgical events. RBCs are currently preserved via hypothermic storage which limits their shelf life to 42 days. Similarly, biobanks of hematopoietic stem cells (HSCs) from umbilical cord blood would be beneficial for regenerative medicine therapies such as HSC transplantations, which offer treatment for blood- and immune-related diseases by reconstituting hematopoiesis. The outcome of these transplantations depends greatly on the quality of the cell product; therefore, it is important for preserved HSCs to have a minimum loss of viability and functionality. The cryopreservation of cells at low sub-zero temperatures (-80 to -196 degC) in a cryoprotectant solution allows for long-term storage. Common cryoprotectants used are 40% glycerol for the cryopreservation of RBCs and 10% dimethyl sulfoxide (DMSO) for the cryopreservation of HSCs. Before clinical use of cryopreserved products, DMSO and glycerol must be removed as they are severely toxic to patients upon infusion. The removal of 40% glycerol from RBCs is complicated, time consuming, and can result in a significant amount of cell damage. DMSO and glycerol also do not address the occurrence of ice recrystallization, which is the main cause of cellular damage during cryopreservation. Ice recrystallization describes the process of ice crystals growing larger and replacing smaller ice crystals, and significantly contributes to the damage of cells post-thaw. Therefore, methods to decrease the concentration of cryoprotectants to improve their removal process while also mitigating ice recrystallization is of interest. In nature, antifreeze proteins and glycoproteins (AF(G)Ps) are found in animals that can survive below-freezing temperatures. The Ben laboratory has used the structural components of AF(G)Ps to design several small-molecule carbohydrates that exhibit ice recrystallization (IRI) activity. O-aryl-b-D-glucosides and N-aryl-D-gluconamides are two classes of IRIs developed that have been used as supplemental additives to DMSO and glycerol to improve the post-thaw viabilities and functionalities of RBCs and HSCs. While many structure-activity relationship studies have been performed amongst these classes, one area of improvement is their solubilities to facilitate their use as cryoprotectants. This thesis focuses on the design of a new class of effective IRIs that have high solubilities (>100 mM in phosphate-buffered saline). Previous studies on the structure of small-molecule IRIs have demonstrated the importance of balancing the hydrophobicity and hydrophilicity within a molecule, making it difficult to achieve high solubilities. This thesis further explores this point by the design and synthesis of IRIs with polar functional groups possessing an overall molecular charge. N-aryl-D-gluconamides bearing amino- and azido-substituents were designed, however their synthesis was unsuccessful. Instead, this work revealed a synthetically facile route towards N-xylo-L-furanosyl amide and ester compounds. Phosphonate-substituted carbohydrates were also designed and synthesized as a means to obtain highly soluble IRIs. All of these compounds displayed high solubilities, however the majority of the compounds exhibited moderate IRI activities. While there are many assays used to measure IRI activity, this thesis also evaluates two of the most common IRI assays and their effect on IRI activity. In addition, the effect that cryoprotective agents (CPAs) like DMSO and glycerol have on IRI activity was also evaluated. In both cases, the type of assay used and the addition of CPAs affected the quantitative values describing IRI activity. Notably, DMSO and glycerol, had an antagonistic effect on the IRI activity of N-aryl-D-gluconamides and antifreeze protein type I. This was a significant observation since these IRIs are sufficient cryoprotectants in the presence of DMSO or glycerol. Lastly in this thesis, phosphonate-substituted IRIs and antifreeze (glyco)proteins (AF(G)Ps) were evaluated as cryoprotectants for the cryopreservation of RBCs and/or HSCs. These studies showed that phosphonate IRIs and AF(G)Ps were not toxic to RBCs and/or HSCs, however the concentrations evaluated were unable to improve the post-thaw viability and/or functionality of these cell types.

No description available.

No description available.

This research investigates the role of ABO blood type of erythrocytes in their dielectrophoretic response. The dielectrophoresis of erythrocytes of positive ABO blood types was studied at 5 V (peak to peak) and 1 MHz frequency AC field. The study revealed that the ABO blood type had an influence on the dielectrophoretic motion of the erythrocytes, particularly separating AB+ and O+ blood types. This is of particular significance since AB+ is a universal acceptor and O+ is a universal donor for blood transfusion purposes. The influence of field parameters, namely field strength and frequency of the AC field, was also studied for erythrocytes of positive ABO blood types. This research revealed that erythrocytes of each blood type respond differently at various frequencies and field strengths.