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UBC Theses and Dissertations

Immunofluorescent flow cytometry in N dimensions : multiplex labelling analysis, and dynamic interpretation of the data Buican, Tudor Nicolae


This thesis investigates two new approaches to the use of multiple antibody labels in flow cytometry. On the experimental side, we develop the Multiplex Labelling method, which allows the number of simultaneous antibody labels to exceed the number of fluorochromes, thus overcoming the technical limitations imposed by the number of available fluorochromes and fluorescence measuring channels. On the theoretical side, we construct a dynamic interpretation of immunofluorescent flow cytometry data, which allows information on the kinetics of cell division and differentiation to be extracted. The first part of the thesis discusses multiplex labelling. Chapter 1.1 presents the theory of this method, a reconstruction formula on which the algorithm for multiplex labelling data processing can be based, and a case study illustrating the use of this method in the triple labelling analysis of murine thymocytes. A murine thymocyte subset not previously described by flow cytometry is observed in this study for the first time. Chapter 1.2 describes the Immunofluorescence Tomograph, a microcomputer-controlled device for the preparation of multiplex labelling solutions. This device makes possible the routine use of multiplex triple labelling, by carrying out a complicated and time consuming part of the experimental protocol. The second part of this thesis deals with the dynamic interpretation of the data. Chapter 2.1 describes the theory of skeletal analysis, which is a coarse topological analysis of immunofluorescent flow cytometry data, concerned with the outlines of regions where the distribution is significantly different' from zero. Chapter 2.2 investigates the finer topological details of the distributions resulting from division and/or differentiation. We show that, under certain reasonable conditions, these distributions acquire simple forms, which can be easily analyzed and compared to actual data. Chapter 2.3 presents murine thymocyte triple labelling data obtained by multiplex analysis. These include data on the embryonic thymus (from day 15 to day 20 of embryonic development), as well as the neonate and adult thymus. The methods developed in chapters 2.1 and 2.2 are applied, and two partial thymocyte lineages are defined. One of these lineages has not been previously reported.

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