Comet assay testing of DNA damage in lymphocyte : a study of stability of cryopreserved cells and effect of pooling of cells from different donors

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Comet assay testing of DNA damage in lymphocyte : a study of stability of cryopreserved cells and effect of pooling of cells from different donors

 

Author: Fok, Sheung Nam Dixon
Title: Comet assay testing of DNA damage in lymphocyte : a study of stability of cryopreserved cells and effect of pooling of cells from different donors
Degree: M.Sc.
Year: 2014
Subject: DNA damage -- Testing.
Lymphocytes -- Testing.
Hong Kong Polytechnic University -- Dissertations
Department: Dept. of Health Technology and Informatics
Pages: ix, 80 leaves : ill. (some col.) ; 30 cm.
Language: English
InnoPac Record: http://library.polyu.edu.hk/record=b2679764
URI: http://theses.lib.polyu.edu.hk/handle/200/7345
Abstract: The comet assay is a commonly used and relatively simple method for detecting DNA damage in single cells. The Fpg-assisted comet assay that measures oxidation-induced lesions in DNA is the version of interest in this study. In human studies, peripheral lymphocytes are the most commonly used cells. For in vitro testing pooled lymphocytes from healthy donors is the commonly used approach. It is useful to test if there is "interaction" between different individual donors' cells in the pooled samples that affects DNA damage. For logistical reasons and to minimize analytical variation, batch testing of cryopreserved cells is done. It is known that lymphocytes can be stored at low temperature for up to 4 weeks without significantly increasing the level of DNA damage. The project aims at testing if lymphocytes can be stored for longer (up to 12 weeks in this project) without significantly increasing the level of DNA damage. In in vitro studies, a single batch of pooled cells is used for the whole experimental series. The stability of pooled cells compared with that of individual cells is of interest. Result showed that no significant difference in terms of DNA damage between individual and pooled cells samples was observed and there was no evidence of a DNA damage-inducing interaction as a result of pooling of cells from individual donors. No significant difference in DNA damage or observable trend was found in those individuals at different time points up to 12 weeks. Also there was no significant DNA damage between pooled cells samples and individual cells samples throughout different time points. The result indicated that cryopreserved cells can be stored for up to 12 weeks without significant effect on DNA damage and this is also true of both single donor and pooled cells. There is also no DNA damaging 'interaction' during storage of pooled cells for up to at least 12 weeks. The findings of this study will be useful in planning and design of studies which use the comet assay as a biomonitoring tool. Pooled lymphocytes can more confidently be used in in vitro studies, and can be stored for an entire experimental series lasting for up to 12 weeks. This will reduce variation from different samples being used, and lower workload from recruiting and harvesting samples. As both single donor and pooled cells can be stored for up to 12 weeks, they can be used as control material in the comet assay, since currently no validated control material exists.

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