Date of Award

1999

Document Type

Master Thesis

Degree Name

Masters of Science (Research)

Department

Biological Sciences

First Advisor

Dr. Héloise Tarrant

Abstract

The aim of this project was to investigate the novel use of the atomic force microscope (AFM) as a technique in the elucidation of leukocyte abnormality. To this end, comparative AFM imaging and elasticity studies were performed on normal and myeloid leukaemic leukocytes.

Initially, an evaluation of techniques for the isolation of specific leukocyte (e.g. lymphocytes, granulocytes, etc.) populations from whole blood or leukocyte concentrates, was carried out. It was found that a silanised glass surface failed to select and immobilise a leukocyte population. However, centrifugation of whole blood in an Isopaque-ficoll gradient solution yielded a viable mixed leukocyte population but the cells were recovered in a physically weakened state and were subsequently broken and damaged during immobilisation in a smear prior to AFM analysis.

In contrast, incubation of whole blood (4°C, 6h), after layering onto a solution of Isopaque-ficoll, yielded a mixed leukocyte population which was sufficiently robust to allow immobilisation in a smear for AFM imaging. Further purification of a lymphocyte population from this leukocyte isolate was achieved by layering the blood onto a polystyrene surface coated with goat anti-human IgG antibodies. The anti-IgG antibodies specifically selected and immobilised lymphocytes to the polystyrene and the non- specifically bound cells were removed by washing.

The Isopaque-ficoll-isolated leukocytes were immobilised on a glass slide for AFM imaging by rapid air-drying of a smear, followed by fixing in 96% (w/v) ethanol. The cells were subsequently imaged by contact mode AFM and a preliminary set of values for the parameters of cell height, width, and surface roughness were obtained for both normal and acute myeloid leukaemia blood samples and for a Jurkat cell line. However, difficulties experienced in defmatively identifying the different leukocyte types during AFM analysis, coupled with low numbers of cells analysed, limited the usefulness of these values when seeking to identify differences between cell types. AFM morphological analysis of the three sample types revealed that the Ip-ficoll-separation had a dehydrating effect on the cells which, in combination with air-drying and ethanol fixation, caused collapse of the nuclei in the cells. This effect rendered the nuclear area visible in AFM images of the Ip-ficoll- isolated cells and was used in this study as a means of putatively identifying cell type.

AFM liquid imaging studies were performed on lymphocytes immobilised on anti- IgG coated polystyrene. Attempts to image the cells in fluid using both contact and non- contact modes proved unsuccessful. The scanning action of the tip caused detachment of the lymphocytes from the polystyrene and so the cells could not be imaged.

The AFM was also used to measure the elasticity of the leukocyte surface. A comparative AFM analysis of the elasticity of normal and chronic myelogenous leukaemia (CML) leukocytes was performed by making force versus indentation (F/I) measurements of the cell surfaces of both sample types. No difference between the estimated mean cell elasticity for Isopaque-ficoll-isolated normal (5.1 ± 1.2 nm/nN) and CML (6.3 ± 0.1 nm/nN) leukocytes was found. In addition, the Isopaque-ficoll separation did not appear to affect the elasticity of the isolated cells. The very low elasticity values obtained were in agreement with previous studies, and were atributed to the dehydrated state of the air-dried cells.

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info:eu-repo/semantics/openAccess

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