Blood

Summary

Blood is a body fluid in the circulatory system of humans and other vertebrates that delivers necessary substances such as nutrients and oxygen to the cells, and transports metabolic waste products away from those same cells. Blood in the circulatory system is also known as peripheral blood, and the blood cells it carries, peripheral blood cells. Blood is composed of blood cells suspended in blood plasma. Plasma, which constitutes 55% of blood fluid, is mostly water (92% by volume), and contains proteins, glucose, mineral ions, hormones, carbon dioxide (plasma being the main medium for excretory product transportation), and blood cells themselves. Albumin is the main protein in plasma, and it functions to regulate the colloidal osmotic pressure of blood. The blood cells are mainly red blood cells (also called RBCs or erythrocytes), white blood cells (also called WBCs or leukocytes), and in mammals platelets (also called thrombocytes). The most abundant cells in vertebrate blood are re

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Virus filtration by size exclusion is a robust means of eliminating viruses in blood or pharma products. Porous polymer membranes with a narrow size distribution are used to allow passage of the protein product but retain viruses. The chosen polymer must resist process-induced physical and chemical wear and is therefore generally inert and hydrophobic material. When filtrating potentially heterogeneous products such as IgG from pooled blood samples, protein adsorption on the polymer surface causes high fouling. This eventually blocks the filter and reduces total filtration volume. A solution to this adsorption problem is to graft a hydrophilic gel-like polymer layer on the membrane inner surface to reduce protein adsorption. The surface properties are thus changed while retaining the stability of the bulk polymer. The grafting was initiated with an electron beam, which creates free radicals by unspecific scission of the polymer backbone through electron irradiation. Two acrylate monomers were used for polymerization, a monofunctional monomer and a bifunctional one, used as a crosslinker to form the gel structure. Two different grafting method were tested. The first grafting method is simultaneous grafting, where the membrane is impregnated with the monomer solution then irradiated. Initiation and polymerization thus occur almost simultaneously. The grafted membrane properties were assessed using flow measurements, protein adsorption tests, FTIR absorbance measurements, virus retention tests and filter integrity tests. Comparing the various grafting approaches, it can be seen that simultaneous grafting lowers considerably protein adsorption, to the price of a highly reduced buffer flow. These filters therefore run slowly but can process more volume before fouling. The sequential grafting method using peroxides as initiators did not show significant filtration improvements compared to the reference membrane when the solution with monomer and crosslinker was used. However, when only monofunctional monomer was used for peroxidation grafting, the protein filtration capacity was increased with limited buffer flow loss. Generally, the sequential method is interesting for graft polymerization of porous membranes because it allows higher degree of grafting and the versatile grafting parameters involved give many development options depending on the application

2010

Development and Characterization of a Microfluidic Chip to Capture Circulating Prostate Cancer Cells

Colette Bichsel

The analysis of circulating tumor cells (CTCs) from the blood of cancer patients promises a better understanding of metastasis formation and tumor phenotypes. CTCs are rare (there is 1 CTC in 1 billion of circulating blood cells) and therefore challenging to isolate. Recently developed highly sensitive microfluidic platforms made a leap forward in trapping these cells efficiently. This work focused on the development of a microfluidic platform that specifically captures prostate cancer cells from a whole blood sample. The versatility of the system was exploited by tethering different capturing antibodies on the microchip surface. Circulating prostate tumor cells were successfully recognized with the microfluidic chip. Furthermore, the platform was enhanced to carry out proliferation assays of captured cells directly on the chip, using hydrogel as a three-dimensional cell culture matrix. Although further improvements regarding the on-chip proliferation assay need to be done, the potential benefit of a functional readout promises new insights in cancer progression that can be translated to advanced cancer staging or prognostic tools.

2011

Microdevice-integrated immunoassays for Point-of-Care Therapeutic Drug Monitoring

Elena-Diana Bojescu

A significant number of drugs exhibit narrow therapeutic windows and high inter-individual pharmacokinetic variability. In such cases, Therapeutic Drug Monitoring (TDM) is compulsory in order to optimize the efficacy of the drug while avoiding adverse effects. Concentration measurements required for dose-adjustments are however complex and are therefore only performed at specialized facilities. The development of a device for accurate whole-blood quantification of critical drugs at the Point-of-Care (POC) would enable fast time-to-result and provide a more convenient solution to patients. This thesis aimed at the design of sample-to-result devices comprising microstructures that allowed for the quantification of small molecules in blood. The designs were based on the development and use of miniaturized fluorescence polarization immunoassays (FPIA). The main feature offered by the FP assay format was the ability to perform homogeneous measurements with no separation or washing steps required. An important result of this thesis is the demonstration that the concentration of small drugs can be quantified in whole blood within paper-like membranes using Fluorescence Polarization Immunoassay (FPIA). Different types of paper-like materials such as glass microfibers, cellulose and filter paper were screened for artefacts such as scattering or autofluorescence. Accurate determination of the fluorescence polarization of red-emitting fluorophores at sub-nanomolar concentrations was found possible within glass fiber membranes. This enabled the development of a competitive immunoassay for the quantification of the antibiotic tobramycin using only 1 ïL of plasma in glass fiber micro-chambers. Furthermore, the same membrane was used for transversal separation of blood cells followed by accurate FPIA read-out at the bottom part of the micro-chamber. Within the therapeutic window, coefficients of variation were around 20% and recoveries between 80-105%, demonstrating the ability to run quantitatively both clinical chemistry and sample preparation by incorporating FPIA in glass fiber membranes. Another important step towards a POC device was the design of a compact, bench-top FP-based analyzer. In this case, glass capillaries were used as detection chambers as they cause little light scattering and for reasons of convenience. Furthermore, the new optical system was employed for tobramycin quantification previously spiked in plasma samples showing good analytical performance in terms of coefficients of variation and recoveries within the therapeutic range of the drug. A more challenging class of drugs are the immunosuppressants, administered in cases of organ transplantation. Here, several fluorescent derivatives of tacrolimus at different wavelengths were synthesized. Their affinity for various biorecognition molecules were tested allowing the choice of a ligand-receptor pair for the development of a FPIA for tacrolimus quantification in buffer. Next, attempts were undertaken to adapt the immunoassay for analysis in whole blood samples. Considerable challenges due to the complexity of the matrix were revealed and initiated efforts to improve the whole blood preparation techniques with the aim of quantifying tacrolimus within its therapeutic range. With the aim of paving the way towards personalized therapies, this thesis makes the realisation of a POC for TDM a realistic goal.

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