Label-free microfluidic enrichment of ring-stage Plasmodium falciparum-infected red blood cells using non-inertial hydrodynamic lift

<b>Background</b> Understanding of malaria pathogenesis caused by Plasmodium falciparum has been greatly deepened since the introduction of in vitro culture system, but the lack of a method to enrich ring-stage parasites remains a technical challenge. Here, a novel way to enrich red blood cells containing parasites in the early ring stage is described and demonstrated.<p></p> <b>Methods</b> A simple, straight polydimethylsiloxane microchannel connected to two syringe pumps for sample injection and two height reservoirs for sample collection is used to enrich red blood cells containing parasites in the early ring stage (8-10 h p.i.). The separation is based on the non-inertial hydrodynamic lift effect, a repulsive cell-wall interaction that enables continuous and label-free separation with deformability as intrinsic marker.<p></p> <b>Results</b> The possibility to enrich red blood cells containing P. falciparum parasites at ring stage with a throughput of ~12,000 cells per hour and an average enrichment factor of 4.3 ± 0.5 is demonstrated.<p></p> <b>Conclusion</b> The method allows for the enrichment of red blood cells early after the invasion by P. falciparumparasites continuously and without any need to label the cells. The approach promises new possibilities to increase the sensitivity of downstream analyses like genomic- or diagnostic tests. The device can be produced as a cheap, disposable chip with mass production technologies and works without expensive peripheral equipment. This makes the approach interesting for the development of new devices for field use in resource poor settings and environments, e.g. with the aim to increase the sensitivity of microscope malaria diagnosis.<p></p&gt

Lift-Effekt auf rote Blutkörperchen und Zellen im Mikrofluss und dessen Anwendung zur Zellsortierung

In dieser Arbeit wurde das Verhalten von roten Blutkörperchen (RBK) im Poiseuille-Fluss unter besonderer Berücksichtigung des trägheitsunabhängigen Lift-Effekts untersucht. Die Ergebnisse zeigen, dass der trägheitsunabhängige Lift-Effekt auf RBK und Blutplättchen (BP) in sehr guter Näherung mit einer für Vesikel entwickelten Theorie beschrieben werden kann. Die Abhängigkeit der Liftgeschwindigkeit von Größe, Form und Deformierbarkeit der Zellen motivierte die Ausnutzung des Lift-Effektes zur Zellsortierung und es wurde die Methode des non-inertial lift-induced cell sorting, kurz NILICS, entwickelt. Das Potential von NILICS zur Sortierung von Zellen nach Größe, Form und Deformierbarkeit wurde anhand der Sortierung zirkulierender Tumorzellen (nach Größe) und Plasmodium falciparum-infizierter RBK (nach Form/Deformierbarkeit) aus RBK-Suspensionen demonstriert. Eine Charakterisierung der Dynamik von RBK im Poiseuille-Fluss zeigte Unterschiede zu den bisher in der Literatur veröffentlichten Beschreibungen selbiger auf. Der Vergleich mit Messungen zur Dynamik von Plasmodium falciparum-infizierten RBK im Poiseuille-Fluss ermöglichte Rückschlüsse auf die durch den Malaria-Parasiten am RBK hervorgerufenen Modifikationen und deren Auswirkungen auf die rheologischen Eigenschaften der infizierten RBK

Lift-Effekt auf rote Blutkörperchen und Zellen im Mikrofluss und dessen Anwendung zur Zellsortierung

In dieser Arbeit wurde das Verhalten von roten Blutkörperchen (RBK) im Poiseuille-Fluss unter besonderer Berücksichtigung des trägheitsunabhängigen Lift-Effekts untersucht. Die Ergebnisse zeigen, dass der trägheitsunabhängige Lift-Effekt auf RBK und Blutplättchen (BP) in sehr guter Näherung mit einer für Vesikel entwickelten Theorie beschrieben werden kann. Die Abhängigkeit der Liftgeschwindigkeit von Größe, Form und Deformierbarkeit der Zellen motivierte die Ausnutzung des Lift-Effektes zur Zellsortierung und es wurde die Methode des non-inertial lift-induced cell sorting, kurz NILICS, entwickelt. Das Potential von NILICS zur Sortierung von Zellen nach Größe, Form und Deformierbarkeit wurde anhand der Sortierung zirkulierender Tumorzellen (nach Größe) und Plasmodium falciparum-infizierter RBK (nach Form/Deformierbarkeit) aus RBK-Suspensionen demonstriert. Eine Charakterisierung der Dynamik von RBK im Poiseuille-Fluss zeigte Unterschiede zu den bisher in der Literatur veröffentlichten Beschreibungen selbiger auf. Der Vergleich mit Messungen zur Dynamik von Plasmodium falciparum-infizierten RBK im Poiseuille-Fluss ermöglichte Rückschlüsse auf die durch den Malaria-Parasiten am RBK hervorgerufenen Modifikationen und deren Auswirkungen auf die rheologischen Eigenschaften der infizierten RBK

Hydrodynamic lift of vesicles and red blood cells in flow — from Fåhræus & Lindqvist to microfluidic cell sorting

Hydrodynamic lift forces acting on cells and particles in fluid flow receive ongoing attention from medicine, mathematics, physics and engineering. The early findings of Fåhræus & Lindqvist on the viscosity change of blood with the diameter of capillaries motivated extensive studies both experimentally and theoretically to illuminate the underlying physics. We review this historical development that led to the discovery of the inertial and non-inertial lift forces and elucidate the origins of these forces that are still not entirely clear. Exploiting microfluidic techniques induced a tremendous amount of new insights especially into the more complex interactions between the flow field and deformable objects like vesicles or red blood cells. We trace the way from the investigation of single cell dynamics to the recent developments of microfluidic techniques for particle and cell sorting using hydrodynamic forces. Such continuous and label-free on-chip cell sorting devices promise to revolutionize medical analyses for personalized point-of-care diagnosis. We present the state-of-the-art of different hydrodynamic lift-based techniques and discuss their advantages and limitations

Hydrodynamic and label-free sorting of circulating tumor cells from whole blood

We demonstrate continuous, passive, and label-free sorting of different in vitrocancercell lines (MV3, MCF7, and HEPG2) as model systems for circulating tumorcells (CTCs) from undiluted whole blood employing the non-inertial lift effect as driving force. This purely viscous, repulsive cell-wall interaction is sensitive to cell size and deformability differences and yields highly efficient cell separation and high enrichment factors. We show that the performance of the device is robust over a large range of blood cell concentrations and flow rates as well as for the different cell lines. The collected samples usually contain more than 90% of the initially injected CTCs and exhibit average enrichment factors of more than 20 for sorting from whole blood samples

Sorting of circulating tumor cells (MV3-melanoma) and red blood cells using non-inertial lift

We demonstrate the method of non-inertial lift induced cell sorting (NILICS), a continuous, passive, and label-free cell sorting approach in a simple single layer microfluidic device at low Reynolds number flow conditions. In the experiments, we exploit the non-inertial lift effect to sort circulating MV3-melanoma cells from red blood cell suspensions at different hematocrits as high as 9%. We analyze the separation process and the influence of hematocrit and volume flow rates. We achieve sorting efficiencies for MV3-cells up to EMV3 = 100% at Hct = 9% and demonstrate cell viability by recultivation of the sorted cells

Interacting dark matter: decay and bremsstrahlung processes

© 2013 Dr. Ahmad Jacob GaleaThough there is substantial indirect astrophysical evidence for the existence of dark matter (DM), it has yet to be directly detected. Consequently, little is known about its internal structure. It is possible that there is a small but finite non-gravitational interaction between dark matter and the Standard Model (SM) which may have observable consequences. The purpose of this thesis is the exploration of some of these interactions and consequences. In particular we consider the possibility that dark matter is unstable on long timescales, as motivated by discrepancies between simulation and observation of structure on sub-galactic scales. We also consider the consequences of electroweak radiative corrections to annihilation processes involving dark matter, as such corrections are necessarily present in many well motivated models. We consider this possibility in the contexts of dark matter annihilation in galactic halos, and production in colliders. Chapter 1 provides an introduction to dark matter, including some of its astrophysical and particle aspects. As a motivation for the following sections, we begin by briefly outlining some of the observational evidence for dark matter. We go on to discuss structure formation, and the cold dark matter distribution on galactic scales. Next we discuss the possibility of non-gravitational interactions involving dark matter, including decay, annihilation, scattering off nuclei, and production. Finally we discuss the determination of the relic abundance in the early Universe, including a discussion of models involving coannihilation. Late decaying dark matter has been proposed as a solution to the small scale structure problems inherent to cold dark matter cosmology. In these models the parent dark matter particle is unstable, and decays into a daughter with near degenerate mass, plus a relativistic final state. In Chapter 2 we review the observational constraints on decaying dark matter, and construct explicit particle physics models to realize this scenario. To achieve this, we introduce a pair of fermionic dark matter candidates and a new scalar field, which obey either a Z4, or a U(1) symmetry. Through the spontaneous breaking of these symmetries, and coupling of the new fields to standard model particles, we demonstrate that the desired decay process may be obtained. We also discuss the dark matter production processes in these models. In Chapter 3 we investigate electroweak radiative corrections to dark matter annihilation into leptons, in which a W or Z boson is also radiated. In many dark matter models the annihilation rate into fermions is helicity suppressed. We demonstrate that bremsstrahlung processes can remove this helicity suppression, causing the branching ratios Br($\ell \nu W$), Br($\ell^+\ell^-Z$), and Br($\bar\nu \nu Z$) to dominate over Br($\ell^+\ell^-$) and Br($\bar\nu \nu$). We find this effect to be most significant in the limit where the dark matter mass is nearly degenerate with the mass of the boson which mediates the annihilation process. Finally, in Chapter 4, we investigate a mono-Z process as a potential dark matter search strategy at the Large Hadron Collider (LHC). In this channel a single Z boson recoils against missing transverse momentum attributed to dark matter particles, $\chi$, which escape the detector. For illustrative purposes we consider the process $q\bar{q} -> \chi\chi Z$ in a toy dark matter model, where the Z boson is emitted from either the initial state quarks, or from the internal propagator. We look for muonic decays of the Z, showing the Standard Model backgrounds to this process to be easily removable with modest selection cuts. We compare signal with Standard Model backgrounds and demonstrate that there exist regions of parameter space where the signal may be clearly visible above background in future LHC data