An automated signal classifier and a semi-automated signal identifier are designed for collecting the dielectrophoretic signatures of cells flowing through a dielectrophoretic cytometer. In past work, the DEP cytometer signals were manually sorted by going through all recorded signals, which is impractical when analyzing 1000’s of cells per day. In the semi-automated method of collection, signals are automatically identified as events and displayed on the user interface to be accepted or rejected by the user. This approach reduced signal collection time by more than half and produced statistics nearly identical to the manual method. The automated signal classifier based on pattern recognition categorizes detection signals as ‘Accept’ or ‘Reject’. Analyzing large volumes of detection signals is possible in much reduced times and may be approaching real time capability.

An automated signal classifier and a semi-automated signal identifier are designed for collecting the dielectrophoretic signatures of cells flowing through a dielectrophoretic cytometer. In past work, the DEP cytometer signals were manually sorted by going through all recorded signals, which is impractical when analyzing 1000’s of cells per day. In the semi-automated method of collection, signals are automatically identified as events and displayed on the user interface to be accepted or rejected by the user. This approach reduced signal collection time by more than half and produced statistics nearly identical to the manual method. The automated signal classifier based on pattern recognition categorizes detection signals as ‘Accept’ or ‘Reject’. Analyzing large volumes of detection signals is possible in much reduced times and may be approaching real time capability.

Stuttgart, Universiẗat, Diss., 2000.

Regensburg, Universiẗat, Diss., 2004.

Berlin, Freie Universiẗat, Diss., 2001. / Dateiformat: zip, Dateien im PDF-Format.

Berlin, Freie Universidad, Diss., 2002. / Dateiformat: zip, Dateien im PDF-Format.

Clausthal, Techn. Universiẗat, Diss., 2004.

Zugl.: Berlin, Freie Univ., Diss., 2001

The detection and identification of particles within fluid samples is key in the prevention of the spreading of disease. This has created a growing need for devices able to successfully separate and identify multiple particles for this purpose while operating at a high enough throughput to be applicable in the field. A well investigated method of manipulating particles in this way is Dielectrophoresis (DEP), which is the use of varied electric fields gradients to generate a force on small particles. The strength of DEP depends of the properties of the particle medium, the signal generating the electric field, and the properties of the particles themselves. This method and its interaction with all small particles, including biological particles such as blood and cancer cells, has allowed devices utilizing this idea to be investigated for various biological purposes. This thesis investigates methods to increase the throughput of these types of devices in order to increase their ability to process large amounts of samples in reasonable amounts of time. This is done in primarily two methods. One approach uses the application of chromatographic methods to DEP devices to separate particles by altering their individual transit time through a device, allowing identification during constant flow. Another method is through mass parallel channels which each individually operate as a standard DEP particle trapping device. This allows for the summation of the maximum flow through the device due to its design layout. / Master of Science / Micrometer scale devices are popular for the identification, separation, and characterization of micron scale particles. This includes uses in biological fields for the manipulation of particles such as blood cells, cancer cells, and bacteria. A common method of manipulating these particles is Dielectrophoresis, a force that causes particles to be repelled or attracted to geometric designs within the device generated by an applied electric field. The strength and direction of this force on the particles is dependent on the properties of the electrical signal applied to the device, the physical properties of the particles, such as size and shape, and the properties of the medium the particles are suspended in within the device. Biological devices utilizing this force have been tested before, allowing for particles to be separated out of mixed particle solutions. Most of these devices operate by moving through very little material at one time, somewhere in the microliter per hour range. This thesis explores attempts to increase the rate at which samples can be processed by these devices in multiple ways. Chapter 2 explores methods of DEP by applying Chromatography principles, which is to constantly move samples through the device at a high rate and slow the target particles, so they exit the device at a different time than other particles. Chapter 3 investigates increasing device throughput by replicating a standard DEP channel multiple times on one device so that several may operate all at once.

Increases in air pollution, especially the particulate component, are associated with increased cardiovascular mortality, possibly through increases in thrombogenic mechanisms. The research presented in this thesis addresses the hypothesis that diesel exhaust particulates (DEP) increase thrombogenicity by impairing the release of tissue plasminogen activator (t-PA) from vascular endothelial cells, thus inhibiting the endogenous fibrinolytic mechanisms that promote thrombus breakdown. The initial aims of this work were to develop an in vivo model of thrombosis, to determine whether exposure to DEP did alter clotting. Initial attempts to develop the Folts’ model (which stimulates thrombus formation via arterial stenosis and mechanical injury), first in male C57/Bl6 mice and later in male Wistar rats, were unsuccessful. An alternative approach, using ferric chloride (FeCl3) to induce chemical injury to the rat carotid artery was found to produce reliable and reproducible thrombotic occlusion: this model was used for all subsequent experiments. The effects of DEP on thrombus formation were assessed in vivo by applying the FeCl3 model. DEP were administered via intratracheal instillation or tail vein injection 2, 6 or 24 hours prior to induction of thrombosis. The effects of DEP were compared with vehicle and suitable controls: carbon black (a clean carbon nanoparticle); quartz (a large non-carbon particle that causes well-characterised pulmonary inflammation). The time to thrombotic occlusion was significantly reduced 6h after intra-pulmonary instillation of DEP (0.5ml of a 1mg/ml suspension). In contrast, instillation of carbon black or quartz had no significant effect on thrombosis, despite causing greater pulmonary (increased neutrophils and levels of interleukin-6, tumour necrosis factor-α and C-reactive protein in bronchoalveolar lavage fluid) and systemic (C-reactive protein in plasma) inflammation than DEP. Direct administration of DEP (0.5mg/kg) to the blood stream resulted in an acute (2 hours after injection) increase in time to thrombotic occlusion in the absence of pulmonary inflammation. A similar (but less pronounced) effect was observed following administration of carbon black (0.5mg/kg). These data suggest that the DEP-mediated increase in thrombosis is independent of pulmonary and systemic inflammation. The mechanisms involved were addressed by measuring platelet-monocyte interactions (flow cytometry) and markers of the endogenous fibrinolytic system (ELISA). Exposure (either instillation of injection) to DEP significantly increased platelet-monocyte aggregation. Carbon black and quartz produced no such effect (but did increase platelet-platelet aggregation). t-PA antigen and activity were reduced, whilst PAI-1 and fibrinogen were increased, following either instillation or injection of DEP. The final aim was to develop a suitable dispersant for use in cell culture to determine whether DEP alter the expression (real-time polymerase chain reaction; rtPCR) and generation (enzyme-linked immunosorbent assay; ELISA) of t-PA and plasminogen activator inhibitor (PAI-1). Cell culture medium containing bovine serum albumin (0.5mg/ml; BSA) provided the best combination for DEP dispersal and maintenance of small particle size (<200nM), without detrimental effects on human umbilical endothelial cells (HUVECs). Exposure (6 and 24 hours) of HUVECs to DEP resulted in reduced basal and thrombin stimulated t-PA and PAI-1 expression. This was mirrored by reduced detection of t-PA and PAI-1 in culture medium. In conclusion, these investigations confirm that exposure to DEP is capable of increasing the rate of thrombus formation and that this is, in part, mediated by an alteration in the endogenous fibrinolytic system. These changes did not appear to be secondary to pulmonary or systemic inflammation. Whilst cell culture experiments suggested DEP could directly alter endogenous fibrinolytic activity in endothelial cells, there was no evidence from these experiments of DEP translocation into the systemic circulation. Thus, this work suggests that DEP is capable of increasing thrombus formation in vivo via several mechanisms. Similar changes may account for the increased thrombus formation in humans exposed to diesel exhaust in air pollution.