Development of Microfluidic Instrumentation for Application in the Diagnosis of Rare Anaemias

Globally, the number of children born every year with a rare anaemia exceeds 500,000. The symptoms of rare anaemias range, depending on the mutation, from mild to severe, and in many cases prove to be fatal. The geographical prevalence of rare anaemias is concentrated in developing countries where resources available for diagnosis and treatment are scarce. The gold standard diagnosis of rare anaemia requires a three-tier investigation which is costly and not readily available in the areas most afflicted. As such, there is a need for a low-cost and user friendly method of diagnosis for these diseases. This thesis investigates the diagnostic abilities of a bio-chemical assay that exposes red blood cells to a low pH shock using microfluidic techniques. This involved the development of a novel low-cost microfluidic instrument, which has been named MeCheM, to run Lab-on-a-Chip devices. The experimental techniques and protocols developed are critically reviewed using healthy blood samples as the control. The results from the control population establish baselines for comparison against the diseased samples. Subsequently, the developed methods are investigated for diagnostic capabilities using rare anaemia blood samples. The results from these investigations suggest that there are observable differences for the developed Flow Test in the case of the Thalassaemia and Hereditary Spherocytosis disorders. Similarly, the developed Cell-Surface Adhesion measurements highlighted significant differences among the Sickle Cell samples. Additionally, secondary investigations indicated correlations between the gold standard Red Blood Cell Count and the RBC Count as measured using MeCheM, and Mean Corpuscular Volume and Average Cell Projected Area (pre-acid addition). The development of MeCheM, a novel microfluidic instrument, as a stand-alone device is a key output from this body of work

Physical properties of red blood cells in aggregation

Red blood cells (RBC) are micron-sized biological objects and the main corpuscular constituent of blood. It flows from larger arteries to very small capillaries. Utilizing a physical approach, this work aims to assess properties that govern blood flows and in particular the disaggregation and aggregation mechanisms of RBC at a single cell level. The interactions of RBCs are thus, investigated experimentally by measuring adhesive forces in the pN range in various model solutions thanks to optical tweezers. While two models for aggregation have been proposed: bridging and depletion, experimental evidence is still lacking to decide which mechanism prevails. The research presented here provides a new insight on the aggregation of RBCs and shows that the two models may not be exclusive. A complete 3-dimensional phase diagram of doublets has been established and confirmed by experiments by varying the adhesive forces and reduced cell volumes. Besides, the effect of aggregation was studied in vitro in a bifurcating microcapillary network and the distribution of aggregates and their stability in such a geometry are reported. Finally, experiments in flow allowed the characterization of the flow field around single RBCs at different velocities. Interesting vortical fluid structures have been also observed thanks to tracer nanoparticles.Rote Blutkörperchen (Erythrozyten) sind biologische Objekte im Mikrometerbereich und der korpuskuläre Hauptbestandteil des Blutes. Es fließt aus größeren Arterien in sehr kleine Kapillaren. Unter Verwendung eines physikalischen Ansatzes zielt diese Arbeit darauf ab, die Eigenschaften zu bewerten, die den Blutfluss und insbesondere die Disaggregations- und Aggregationsmechanismen der RBC auf Einzelzellebene regeln. Die Interaktionen der Erythrozyten werden daher experimentell untersucht, indem Adhäsionskräfte im pN-Bereich in verschiedenen Modelllösungen mit Hilfe einer optischen Pinzette gemessen werden. Während mit Bridging und Depletion zwei Modelle für die Aggregation vorgeschlagen wurden, fehlen noch experimentelle Beweise, um zu entscheiden, welcher Mechanismus vorherrscht. Die hier vorgestellte Forschung liefert neue Erkenntnisse über die Aggregation von RBCs und zeigt, dass die beiden Modelle möglicherweise nicht exklusiv sind. Es wurde ein vollständiges dreidimensionales Phasendiagramm von Dubletten erstellt und experimentell durch Variation der Adhäsionskräfte und reduzierte Zellvolumina bestätigt. Außerdem wurde der Effekt der Aggregation in vitro in einem sich gabelförmigen Mikrokapillarnetz untersucht, und es wird über die Verteilung der Aggregate und ihre Stabilität in einer solchen Geometrie berichtet. Schließlich erlaubten Strömungsexperimente die Charakterisierung des Strömungsfeldes um einzelne RBCs bei unterschiedlichen Geschwindigkeiten. Dank Tracer-Nanopartikeln konnten auch interessante wirbelartige Fluidstrukturen beobachtet werden

The dynamics of a capsule in a wall-bounded oscillating shear flow

The motion of an initially spherical capsule in a wall-bounded oscillating shear flow is investigated via an accelerated boundary integral implementation. The neo-Hookean model is used as the constitutive law of the capsule membrane. The maximum wall-normal migration is observed when the oscillation period of the imposed shear is of the order of the relaxation time of the elastic membrane; hence, the optimal capillary number scales with the inverse of the oscillation frequency and the ratio agrees well with the theoretical prediction in the limit of high-frequency oscillation. The migration velocity decreases monotonically with the frequency of the applied shear and the capsule-wall distance. We report a significant correlation between the capsule lateral migration and the normal stress difference induced in the flow. The periodic variation of the capsule deformation is roughly in phase with that of the migration velocity and normal stress difference, with twice the frequency of the imposed shear. The maximum deformation increases linearly with the membrane elasticity before reaching a plateau at higher capillary numbers when the deformation is limited by the time over which shear is applied in the same direction and not by the membrane deformability. The maximum membrane deformation scales as the distance to the wall to the power 1/3 as observed for capsules and droplets in near-wall steady shear flows

Isolation of Circulating Tumor Cells with Acoustophoresis : Towards a biomarker assay for prostate cancer

Microfluidics has emerged as an essential approach in the development of novel technological platforms to detect and isolate rare circulating tumor cells (CTCs) in the blood of cancer patients. Micro-scaled fluidic systems offer means to precisely control fluid flow. This enables cell separation when combined with techniques for manipulating cells across fluid streams. Various microfluidic methods have been developed, either using passively generated forces or an applied force field methodology to move cells across streamlines. Acoustophoresis uses ultrasonic standing waves to separate cells and particles in microfluidic channels. An acoustic standing wave field generates acoustic forces that acts on cells and particles based on their individual acoustic properties and forms the basis for the cell separation technology explored in this thesis. In this dissertation, a novel approach for live CTC isolation has been developed. Micron-sized elastomeric particles with negative acoustic contrast were used for negative selection acoustophoresis. The surface of the elastomeric particles was functionalized to bind WBCs through the CD45 antigen, which enabled their transportation to pressure antinodes and facilitated an enrichment of cancer cells at the pressure node. Live cell negative selection acoustophoresis was demonstrated as a proof-of-concept study in paper 1 and was further extended to carry out processing of whole blood by a two-step acoustophoresis method in paper 2. The sample throughput is an important parameter for microfluidic processing of clinical samples, especially for rare cell applications where a larger volume might be required for the detection of target cells. In paper 3, a fluid inertia phenomenon that may compromise cell separation performance at higher flow velocities was discovered. The inertial effects in an acoustofluidic device at increased sample throughputs and its consequences on particle separations were therefore investigated. Through numerical modelling and experimental validation, the main reason for the impaired acoustophoresis separation, at elevated flow rates, was attributed to the formation of a curved fluid boundary between the sample and sheath flow, both at the inlet of the separation channel as well as at the outlet flow splitter.Finally, paper 4 outlines the benchmarking of CTC-acoustophoresis to the FDA cleared CELLSEARCH system in a comparative clinical study. Higher numbers of CTCs were detected after acoustophoretic processing of the patient samples as compared to the CELLSEARCH system. Further studies are currently being conducted to establish the full performance characteristics of CTC-acoustophoresis in the laboratory setting.To conclude, the presented dissertation extends the use of acoustophoresis towards the clinical application of CTC enrichment of live and fixed cells. The aim of establishing an efficient technology that can target the full heterogeneity of the rare tumor cells is essential for the development of novel CTC biomarker assays for metastatic cancer. This dissertation builds towards the goal of an unbiased CTC isolation approach

UTILIZING DIELECTROPHORESIS TO DETERMINE THE PHYSIOLOGICAL DIFFERENCES OF EUKARYOTIC CELLS

Type 1 diabetes affects over 108,000 children, and this number is steadily increasing. Current insulin therapies help manage the disease but are not a cure. Over a child’s lifetime they can develop kidney disease, blindness, cardiovascular disease and many other issues due to the complications of type 1 diabetes. This autoimmune disease destroys beta cells located in the pancreas, which are used to regulate glucose levels in the body. Because there is no cure and many children are affected by the disease there is a need for alternative therapeutic options that can lead to a cure. Human mesenchymal stem cells (hMSCs) are an important cell source for stem cell therapeutics due to their differentiation capacity, self-renewal, and trophic activity. hMSCs are readily available in the bone marrow, and act as an internal repair system within the body, and they have been shown to differentiate into insulin producing cells. However, after isolation hMSCs are a heterogeneous cell population, which requires secondary processing. To resolve the heterogeneity issue hMSCs are separated using fluorescent- and magnetic-activate cell sorting with antigen labeling. These techniques are efficient but reduce cell viability after separation due to the cell labeling. Therefore, to make hMSCs more readily available for type 1 diabetes therapeutics, they should be separated without diminishing there functional capabilities. Dielectrophoresis is an alternative separation technique that has the capability to separated hMSCs. This dissertation uses dielectrophoresis to characterize the dielectric properties of hMSCs. The goal is to use hMSCs dielectric signature as a separation criteria rather than the antigen labeling implemented with FACS and MACS. DEP has been used to characterize other cell systems, and is a viable separation technique for hMSCs

Fabrication of Micro- and Nano-Fluidic Lab-On-A-Chip Devices Utilizing Proton Beam Writing Technique

Ph.DDOCTOR OF PHILOSOPH