Microfluidics in Haemostasis: a Review

Haemostatic disorders are both complex and costly in relation to both their treatment and subsequent management. As leading causes of mortality worldwide, there is an ever-increasing drive to improve the diagnosis and prevention of haemostatic disorders. The field of microfluidic and Lab on a Chip (LOC) technologies is rapidly advancing and the important role of miniaturised diagnostics is becoming more evident in the healthcare system, with particular importance in near patient testing (NPT) and point of care (POC) settings. Microfluidic technologies present innovative solutions to diagnostic and clinical challenges which have the knock-on effect of improving health care and quality of life. In this review, both advanced microfluidic devices (R&D) and commercially available devices for the diagnosis and monitoring of haemostasis-related disorders and antithrombotic therapies, respectively, are discussed. Innovative design specifications, fabrication techniques, and modes of detection in addition to the materials used in developing micro-channels are reviewed in the context of application to the field of haemostasi

A Novel Microbial Source Tracking DNA Microarray Used for Pathogen Detection in Environmental Systems

Pathogen detection and the identification of fecal contamination sources can be challenging in environmental and engineered treatment systems. Factors including pathogen diversity and ubiquity of fecal indicator bacteria hamper risk assessment and remediation of contamination sources. Therefore, a quick method that can detect and identify waterborne pathogens in environmental systems is needed. In this work, a custom microarray targeting pathogens (viruses, bacteria, protozoa), microbial source tracking (MST) markers, mitochondria DNA (mtDNA) and antibiotic resistance genes was used to detect over 430 selected gene targets in whole genome amplification (WGA) DNA and complementary DNA (cDNA) isolated from sewage and animal (avian, cattle, poultry and swine) feces, freshwater and marine water samples, sewage spiked surface water samples, treated wastewater and sewage contaminated produce.;A combination of perfect match and mismatch probes on the microarray reduced the likelihood of false positive detections, thus increasing the specificity of the microarray for various gene targets. A linear decrease in fluorescence of positive probes over a 1:10 dilution series demonstrated a semi-quantitative relationship between gene concentrations in a sample and microarray fluorescence. Various pathogens, including norovirus, Campylobacter fetus, Helicobacter pylori, Salmonella enterica, and Giardia lamblia were detected in sewage via the microarray, as well as MST markers and resistance genes to aminoglycosides, beta-lactams, and tetracycline. Sensitivity (percentage true positives) of MST results in sewage and animal waste samples (21--33%) was lower than specificity (83--90%, percentage of true negatives). Next generation sequencing (NGS) of DNA from the fecal samples revealed two dominant bacterial families that were common to all sample types: Ruminococcaceae and Lachnospiraceae. Five dominant phyla and 15 dominant families comprised 97% and 74%, respectively, of sequences from all fecal sources.;Waterborne pathogens were also detectable via the microarray in freshwater, marine water and sewage spiked surface water samples as well as treated wastewater. Ultrafiltration was used to concentrate microorganisms (bacteria, viruses, protozoa and parasites) from several liters of environmental and treated water samples. Dead-end ultrafiltration (DEUF) was shown to have a 61.4 +/- 47.8 % recovery efficiency and 46-fold concentration increasing ability. Then WGA was utilized to increase gene copies and lower the microarray detection limit. Viruses, including adenovirus, bocavirus, Hepatitis A virus, and polyomavirus were detected in human associated water samples as well as pathogens like Legionella pneumophila, Shigella flexneri, C. fetus and genes coding for resistance to aminoglycosides, beta-lactams, tetracycline. Microbial source tracking results indicate that sewage spiked freshwater and marine samples clustered separately from other fecal sources including wild and domestic animals via non-metric dimensional scaling. A linear relationship between qPCR and microarray fluorescence was found, indicating the semi-quantitative nature of the MST microarray.;Multiple displacement amplification (MDA), which is an important type of WGA, is a widely used tool to amplify genomic nucleic acids. The strong amplification efficiency of MDA and low initial template requirement make MDA an attractive method for environmental molecular and NGS studies. However, like other nucleic acid amplification techniques, various factors may influence MDA efficiency including template concentration (e.g. rare species swamping out), GC amplification bias and genome length favoring amplification of longer genomes. It was found that MDA increased nucleic acids in mixed environmental samples approximately 4.24 +/- 1.40 (log, average +/- standard deviation) for 16S rRNA gene of Enterococcus faecalis, 1.90 +/- 1.70 for RNA polymerase gene of human norovirus, 8.83 +/- 2.88 for T antigen gene of human polyomavirus, 3.83 +/- 0.93 for uidA gene of Escherichia coli, 4.96 +/- 0.32 for invA gene of S. enterica and 8.77 +/- 2.85 for 16S rRNA gene of human Bacteroidales. The template length, concentration and GC content were found to influence MDA efficiency. The results mainly show that the MDA will be more efficient the longer the template length, the greater the initial concentration of nucleic acids and the lower the GC content of the template.;Overall, the results of this work show that 1) the microarray and sample handling technique is suitable for pathogen detection from feces and sewage; 2) when combined with ultrafiltration techniques, the microarray can also be used as a pathogen detection tool in environmental waters; 3) template length, and initial concentration increase MDA efficiency, but higher GC content template negatively effects MDA efficiency. The proposed microarray can be used for pathogen detection in feces, wastewater treatment plant sewage, treated wastewater and environmental waters. Further the proposed method is potentially applicable to pathogen/microorganism detections on vegetables, seafood, in hospital settings, industrial wastewater, and aquaculture settings

ENDOTHELIAL PROGENITOR CELL RECRUITMENT IN A WOUND HEALING MICROFLUIDIC VASCULAR MODEL

During wound healing, endothelial progenitor cells (EPCs) are recruited from the bone marrow and directed to the site of injury. At the site of injury, hypoxic conditions promote TNF-α, which up regulates intercellular adhesion molecule-1 (ICAM-1). EPCs use ICAM-1 to attach to endothelial cells (ECs) lining blood vessels. Here, we design, develop and test a three-dimensional microbioreactor system (3-D MBR) with precise control and monitoring of oxygen and media flow rate. We first analyze the transport of oxygen in the proposed device. Following fabrication of the 3-D MBR, we next utilized a step-wise seeding technique, which resulted in confluency of human umbilical vein endothelial cells (HUVECs) on all four sides of the device. We next examine endothelial colony forming cell (ECFC) attachment and retention onto HUVECs using conventional 2-D cultures. HUVECs are pre-stimulated with one of four conditions: 21% oxygen (atmospheric), atmospheric with TNF-α-supplemented media, 1% oxygen (hypoxia), and lastly hypoxia with TNF-α-supplemented media. We show the highest attachment and retention of ECFCs on HUVECs pre-treated with TNF-α and 1% oxygen, which correlated with the highest expression levels of ICAM-1. Using the new 3-D MBR system we next demonstrate that TNF-α and hypoxia, when used in conjunction, significantly increase EPC attachment on ECs under pathologically relevant flow conditions. The 3-D MBR system allows us to mimic the oxygen and shear stress environment in the vasculature, thus providing a step between traditional in-vitro and in-vivo experimentation to model a variety of vascular-related disorders, especially wound healing

Advanced Applications of Rapid Prototyping Technology in Modern Engineering

Rapid prototyping (RP) technology has been widely known and appreciated due to its flexible and customized manufacturing capabilities. The widely studied RP techniques include stereolithography apparatus (SLA), selective laser sintering (SLS), three-dimensional printing (3DP), fused deposition modeling (FDM), 3D plotting, solid ground curing (SGC), multiphase jet solidification (MJS), laminated object manufacturing (LOM). Different techniques are associated with different materials and/or processing principles and thus are devoted to specific applications. RP technology has no longer been only for prototype building rather has been extended for real industrial manufacturing solutions. Today, the RP technology has contributed to almost all engineering areas that include mechanical, materials, industrial, aerospace, electrical and most recently biomedical engineering. This book aims to present the advanced development of RP technologies in various engineering areas as the solutions to the real world engineering problems

Age-related macular degeneration: interventional tissue engineering and predictive modeling of disease progression

Thesis (Ph.D.)--Boston UniversityAge-related macular degeneration (AMD) is the leading cause of irreversible blindness in people over the age of 50. As many as 50 million people are affected by AMD worldwide and prevalence is expected to continue to rise due to an aging population. There are two forms of the disease, dry (geographic atrophy) and wet (choroidal neovascularization), both of which result in retinal degeneration and central vision loss. Although anti-vascular endothelial growth factor therapies are moderately successful at treating the wet form, there are no treatments currently available for the more common dry form. Pharmacological therapies have been extensively explored for the treatment of dry AMD, but have achieved little success because the pathogenesis underlying AMD is unknown and likely varies among patients . Recently, tissue engineering has emerged as a promising approach to restore function by replacing diseased retinal tissue with healthy retinal pigment epithelium (RPE). While AMD-associated vision loss occurs when photoreceptors degenerate, this process arises as a consequence of earlier RPE dysfunction. In the healthy retina, the RPE acts as a critical regulator of the microenvironment for both photoreceptors and the nearby vasculature. However in AMD, the RPE no longer performs these essential homeostatic functions leading to photoreceptor apoptosis and vision loss. This dissertation describes the development and in vitro characterization of a tissue engineering scaffold for RPE delivery as potential treatment for dry AMD. First, a novel microfabrication-based method termed "pore casting" was developed to produce thin scaffolds with highly controlled pore size, shape, and spacing. Next, human RPE were cultured on pore-cast poly(c-caprolactone) (PCL) scaffolds and compared to cells on track-etched polyester, the standard RPE culture substrate. RPE on porous PCL demonstrated enhanced maturation and function compared to track-etched polyester including improved pigmentation, barrier formation, gene expression, growth factor secretion, and phagocytic degradation. Lastly, this study established a patient-specific method for predicting AMD progression using retinal oxygen concentration. This approach differs from current diagnosis techniques because it uses physiologically-relevant mechanisms rather than generalized clinical associations which have little, if any, prognostic value

Effects of complex vessel geometries on neutrophil margination and adhesion in post-capillary venules

The inflammatory process is a regular occurrence within a healthy body. As part of the inflammatory process, leukocytes flow through blood vessels and are recruited to the region of the injury. Neutrophils play a significant role in this process; however the margination of neutrophils to particular locations in micro vessels is not fully understood. Post capillary venules, in particular, have complex geometries which may contribute to non-uniform adhesion of neutrophils. Margination is a phenomenon that occurs during the relatively early phases of inflammation; as a result of dilation of capillaries and slowing of the bloodstream, leukocytes tend to occupy the periphery of the cross-sectional lumen. Other investigations have looked at the adhesion of neutrophils in vivo or flow patterns in converging tubes, but the correlation between flow patterns in complex geometries and neutrophil margination is not well understood. This study seeks to investigate correlations between margination and bulk flow patterns as well as parameters that affect bulk flow properties. The primary aim of this investigation is to create specific computational and in vitro models based on in vivo data that isolate the hydrodynamic mechanisms associated with complex geometries. Main geometric factors that were investigated were surface roughness, branch geometries, number of convergences and squared vs. rounded t-junctions. To determine the effect of surface roughness a large scale parallel plate flow chamber model as well as a microfabrication technique to simulate roughness at the blood vessel scale were created that simulate surface roughness due to endothelial cell nuclei. CFD modeling was also used to determine effects of other geometric factors including branch geometries, number of convergences and squared vs. rounded t-junctions. Overall, results from this study suggest that complex geometries can have a significant role on neutrophil margination and adhesion in blood vessels. A preliminary relationship between wall shear stress and margination was established

A Microfluidic Approach for Investigating the Role of Intrathrombus Transport in Thrombosis and Hemostasis

Biological and physical factors interact to modulate blood response in a wounded vessel, resulting in a hemostatic clot or an occlusive thrombus. Wall shear stress (τw) and pressure differential (ΔP) across the wound from the lumen to the extravascular compartment may impact hemostasis. This thesis describes the design of a microfluidic device capable of flowing human blood over a side channel plugged with collagen (± tissue factor) while independently controlling ΔP and τw. Using this device we were able to investigate the impact of physiologic hemodynamics on the growth and architecture of human blood clots. Our results revealed that both wall shear rate and the transthrombus pressure gradient govern clot development leading to the formation of two distinct intrathrombus regions; a core of highly-activated platelets and fibrin covered by a shell of less-activated platelets. These regions mimic the activation gradients of clots formed in vivo. We demonstrated that core development was dependent on the transthrombus pressure gradient restricting thrombin localization while shell development was dependent on wall shear rates. We also found that fibrin polymerization inhibited thrombin activity at both arterial and venous shear rates. However, the mechanism of this inhibition is shear dependent. At venous shears thrombin activity is inhibited by γ\u27-fibrin(ogen) binding. While at arterial and pathological shear rates the clot forms a more dense structure leading to physical trapping of thrombin independent of γ\u27-fibrin(ogen) binding. Taken together our data supports a model where clot architecture is maintained under various conditions by shear-specific thrombin inhibition mechanisms. Lastly, we demonstrated that the prevailing hemodynamics dilute ADP and thromboxane to regulate platelet contractility, a newly defined flow sensing mechanism to regulate clot function. The field of in vitro hemostasis and thrombosis research has lacked an assay capable of independently studying the effects of ΔP and local τw on clot development and function. Our microfluidic device bridges this gap, while providing new insights into the mechanisms of hemostasis and thrombosis, where hemostatic clot development must balance both thrombotic and hemorrhagic risks in order to rapidly and controllably cease bleeding