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Cytogenetic evaluation of human bone marrow cells is one of the principal sources of diagnostic and prognostic information in the evaluation of the myeloid leukemias. In the majority of cases, these diseases are characterized by non-random chromosomal changes in the cells of the malignant clone. The chromosomal abnormalities are present only in the leukemic cells, which are distributed along with normal cells in the bone marrow and throughout the circulation. The objective of this thesis was to test the hypothesis that suitable criteria could be established for automated metaphase detection using human bone marrow preparations. This involved computerized, low resolution scanning of a specimen slide, and the measurement of object features which allowed metaphases to be adequately distinguished from nuclei and debris. Two approaches were investigated. The first used a line-scanning system, in which microscope slides were scanned line by line with a linear CCD detector, and focussing was performed automatically. Eighteen signal features were measured for each detected object. Three group discriminant function analysis was performed on objects from a large number of slides from both types of preparations, in order to distinguish metaphases from nuclei and debris. The second method evaluated the use of a frame scanning system. Objects were detected in a frame-by-frame scan of microscope slides, using a two dimensional CD camera. Feature measurements were performed for all objects within a specified area range, and three group discriminant function analysis was performed on data from a large number of slides. In both approaches, the performance of the discriminant functions was evaluated on independent samples collected from a number of patients, in order to determine the operational error rates of the systems. The sensitivity of the line scan system for metaphase detection was 86%, compared to 92% fror the frame scannning system, while the specificity was 84% for the line scan system, and 86% for the frame scanning system. The frame scan system was shown to be useful for determining the mitotic index of cells cultured for varying periods of time prior to fixation. Four patients with AML were examined, and the results of the analysis show that the mitotic indices could be determined in this way to an accuracy of approximately 5%. The mitotic indices differed as a function of time for different patients. / Science, Faculty of / Physics and Astronomy, Department of / Graduate

Optical microscopy is the simplest and the gold standard method adopted for the screening and subsequent diagnosis of various hematological and infectious diseases like malaria, sickle cell disease, tuberculosis etc. In addition to infectious disease diagnosis, its applications range from routine blood tests to the more sophisticated cancer biopsy sample analysis. Microscopy Tests (MTs) follow a common procedural workflow: (1) A technician prepares a smear of the given sample on a glass slide in a specific manner depending on the sample and the disease to be diagnosed; (2) The smeared slide is subsequently exposed to fixative agents and different histochemical stains specific to the diagnosis to be performed and (3) the prepared slide is then observed under a high quality bright- field bench-top microscope. An expert pathologist/cytologist is required to manually examine multiple fields-of-views of the prepared slide under appropriate magnification. Multiple re-adjustments in the focus and magnification makes the process of microscopic examination time consuming and tedious. Further, the manual intervention required in all the aforementioned steps involved in a typical MT, makes it inaccessible to rural/resource limited conditions and restricts the diagnostics to be performed by trained personnel in laboratory settings. To overcome these limitations, there has been considerable research interest in developing cost-effective systems that help in automating MTs. The work done in this thesis addresses these issues and proposes a two-step solution to the problem of affordable automation of MTs for cellular imaging and subsequent diagnostic assessment. The first step deals with the development of a low cost portable system that employs custom-built microscopy setup using o -the-shelf optical components, low cost motorized stage and camera modules to facilitate slide scanning and digital image acquisition. It incorporates a novel computational approach to generate good quality in-focus images, without the need for employing high-end precision translational stages, thereby reducing the overall system cost. The process of slide analysis for result generation is further automated by using image analysis and classification algorithms. The application of the developed platform in automating slide based quantitative detection of malaria is reported in this thesis. The second aspect of the thesis addresses the automation of slide preparation. A major factor that could influence the analysis results is the quality of the prepared smears. The feasibility of automating and standardizing the process of slide preparation using Microfluidics with appropriate surface fictionalization is explored and is demonstrated in the context of automated semen analysis. As an alternative to the mechanism of fixing the spermatozoa to the glass slide by smearing and chemical treatment with fixative, microfluidic chips pre-coated with adhesive protein are employed to capture and immobilize the cells. The subsequent histochemical staining is achieved by pumping the stains through the microfluidic device. The proof-of-principle experiments performed in this thesis demonstrate the feasibility of the developed system to provide an end-to-end cost-effective alternative solution to conventional MTs. This can further serve as an assistive tool for the pathologist or in some cases completely eliminate the manual intervention required in MTs enabling repeatability and reliability in diagnosis for clinical decision making