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.

Construction and detectors’ characterization of a new detection system for FAMU Experiment

From 2010, the Proton Radius has become one of the most interest value to determine. The first proof of not complete understanding of its internal structure was the measurement of the Lamb Shift using the muonic hydrogen, leading to a value 7σ lower. A new road so was open and the Proton Radius Puzzle epoch begun. FAMU Experiment is a project that tries to give an answer to this Puzzle implementing high precision experimental apparatus. The work of this thesis is based on the study, construction and first characterization of a new detection system. Thanks to the previous experiments and simulations, this apparatus is composed by 17 detectors positioned on a semicircular crown with the related electronic circuit. The detectors' characterization is based on the use of a LabView program controlling a digital potentiometer and on other two analog potentiometers, all three used to set the amplitude of each detector to a predefined value, around 1.2 V, set on the oscilloscope by which is possible to observe the signal. This is the requirement in order to have, in the final measurement, a single high peak given by the sum of all the signals coming from the detectors. Each signal has been acquired for almost half of an hour, but the entire circuit has been maintained active for more time to observe its capacity to work for longer periods. The principal results of this thesis are given by the spectra of 12 detectors and the corresponding values of Voltages, FWHM and Resolution. The outcomes of the acquisitions show also another expected behavior: the strong dependence of the detectors from the temperature, demonstrating that an its change causes fluctuations in the signal. In turn, these fluctuations will affect the spectrum, resulting in a shifting of the curve and a lower Resolution. On the other hand, a measurement performed in stable conditions will lead to accordance between the nominal and experimental measurements, as for the detectors 10, 11 and 12 of our system.