Optimisation of chemical lysis protocol for point-of-care leukocyte differentiation

The full blood cell (FBC) count is the most common indicator of diseases. At present hematology analyzers are used for the blood cell characterization, but, recently, there has been interest in using techniques that take advantage of microscale devices and intrinsic properties of cells for increased automation and decreased cost. Microfluidic technologies offer solutions to handling and processing small volumes of blood (2-50 uL taken by finger prick) for point-of-care(PoC) applications. Several PoC blood analyzers are in use and may have applications in the fields of telemedicine, out patient monitoring and medical care in resource limited settings. They have the advantage to be easy to move and much cheaper than traditional analyzers, which require bulky instruments and consume large amount of reagents. The development of miniaturized point-of-care diagnostic tests may be enabled by chip-based technologies for cell separation and sorting. Many current diagnostic tests depend on fractionated blood components: plasma, red blood cells (RBCs), white blood cells (WBCs), and platelets. Specifically, white blood cell differentiation and counting provide valuable information for diagnostic purposes. For example, a low number of WBCs, called leukopenia, may be an indicator of bone marrow deficiency or failure, collagen- vascular diseases, disease of the liver or spleen. The leukocytosis, a high number of WBCs, may be due to anemia, infectious diseases, leukemia or tissue damage. In the laboratory of hybrid biodevices, at the University of Southampton,it was developed a functioning micro impedance cytometer technology for WBC differentiation and counting. It is capable to classify cells and particles on the base of their dielectric properties, in addition to their size, without the need of labeling, in a flow format similar to that of a traditional flow cytometer. It was demonstrated that the micro impedance cytometer system can detect and differentiate monocytes, neutrophils and lymphocytes, which are the three major human leukocyte populations. The simplicity and portability of the microfluidic impedance chip offer a range of potential applications in cell analysis including point-of-care diagnostic systems. The microfluidic device has been integrated into a sample preparation cartridge that semi-automatically performs erythrocyte lysis before leukocyte analysis. Generally erythrocytes are manually lysed according to a specific chemical lysis protocol, but this process has been automated in the cartridge. In this research work the chemical lysis protocol, defined in the patent US 5155044 A, was optimized in order to improve white blood cell differentiation and count performed by the integrated cartridge.

Automatic Design of Boolean Networks for Modelling Differentiation Trees

Real living cell is a complex system governed by many process which are not yet fully understood: the process of cell differentiation is one of these. In this thesis work we make use of a cell differentiation model to develop gene regulatory networks (Boolean networks) with desired differentiation dynamics. To accomplish this task we have introduced techniques of automatic design and we have performed experiments using various differentiation trees. The results obtained have shown that the developed algorithms, except the Random algorithm, are able to generate Boolean networks with interesting differentiation dynamics. Moreover, we have presented some possible future applications and developments of the cell differentiation model in robotics and in medical research. Understanding the mechanisms involved in biological cells can gives us the possibility to explain some not yet understood dangerous disease, i.e the cancer. Le cellula è un sistema complesso governato da molti processi ancora non pienamente compresi: il differenziamento cellulare è uno di questi. In questa tesi utilizziamo un modello di differenziamento cellulare per sviluppare reti di regolazione genica (reti Booleane) con dinamiche di differenziamento desiderate. Per svolgere questo compito abbiamo introdotto tecniche di progettazione automatica e abbiamo eseguito esperimenti utilizzando vari alberi di differenziamento. I risultati ottenuti hanno mostrato che gli algoritmi sviluppati, eccetto l'algoritmo Random, sono in grado di poter generare reti Booleane con dinamiche di differenziamento interessanti. Inoltre, abbiamo presentato alcune possibili applicazioni e sviluppi futuri del modello di differenziamento in robotica e nella ricerca medica. Capire i meccanismi alla base del funzionamento cellulare può fornirci la possibilità di spiegare patologie ancora oggi non comprese, come il cancro.

Finite element modeling of flow/compression-induced deformation of alginate scaffolds for bone tissue engineering

Trauma or degenerative diseases such as osteonecrosis may determine bone loss whose recover is promised by a "tissue engineering“ approach. This strategy involves the use of stem cells, grown onboard of adequate biocompatible/bioreabsorbable hosting templates (usually defined as scaffolds) and cultured in specific dynamic environments afforded by differentiation-inducing actuators (usually defined as bioreactors) to produce implantable tissue constructs. The purpose of this thesis is to evaluate, by finite element modeling of flow/compression-induced deformation, alginate scaffolds intended for bone tissue engineering. This work was conducted at the Biomechanics Laboratory of the Institute of Biomedical and Neural Engineering of the Reykjavik University of Iceland. In this respect, Comsol Multiphysics 5.1 simulations were carried out to approximate the loads over alginate 3D matrices under perfusion, compression and perfusion+compression, when varyingalginate pore size and flow/compression regimen. The results of the simulations show that the shear forces in the matrix of the scaffold increase coherently with the increase in flow and load, and decrease with the increase of the pore size. Flow and load rates suggested for proper osteogenic cell differentiation are reported.