Micronucleus cytome assay in buccal cells – Method development


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A Master's thesis.
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Master (thesis) (master)
Micronucleus cytome assay in buccal cells – Method development
DANUSER Brigitta
HOPF Nancy
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Université de Lausanne, Faculté de biologie et médecine
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Our aim is to develop a buccal cell micronucleus (MN) assay relying on automated image analysis, with the MetaSystems Metafer image cytometry system. A prerequisite for this objective is a well-optimized slide preparation protocol. Our objectives were to: evaluate samples stored for 5, 10 and 10 days at temperatures: room temperature (RT), 4°, and -20°C. Moreover, we wanted to assess effectiveness of self-collection by participants using the number of cells harvested as determinant, and finally, test a particular DNA stain, known as DAPI, in the automated system.
After ethics committee approval, we recruited 36 healthy participants aged 19 to 30. Buccal cell collection with a cytobrush was performed by participants themselves (self- collection), following given written instructions. The cells were extracted from the cytobrushes by gently shaking them in Saccomanno’s solution (10ml). This solution was split into two identical filled (5ml) tubes. One tube was used for direct slide preparation (control) and the second was stored for 5, 10 or 20 days either at -20°, 4° or RT before processing. Slides were prepared following a specific and adapted protocol, DAPI stain was applied, and slides were reviewed by fluorescence microscope. During microscope analysis, each slide was cautiously examined by the researcher, and two images were taken at x2.52 magnification at distinct regions. Cell counting was performed on both images and we regarded the sum for comparison between slide cell quantities. Each experimental value was paired with their respective control by computing experimental value/control value ratios (Exp/Ctrl). Each slide was subjectively given a score by the same researcher depending on sampling quality from their associated participant: 1 for poor quality; 2 mediocre quality; and 3 for good quality.
In total, 12,240 cells were counted, with a mean value of 170 cells per slide. At 5 and 10 days, independent of storage temperature, mean ratios were all greater than 100%. This tendency was reversed after 15 days. Indeed, 10 out of the 12 stored samples (83%) had lower cell counts than their respective controls. Participants with the lowest score had a sample mean of 31 cells versus 185 and 246 from those who received 2 and 3, respectively. Finally, male participants had greater cell pools than females with a mean of 180 versus 123 cells.
These results suggest that cells can be stored at room temperature up to 10 days. Consequently, slide quality would not be affected by temperature if participants collected their samples at home and directly sent them by secure post. However, results discourage cell storage for more than 10 days, specifically at room temperature, for which an important decrease of cell count was observed. Cell number was correlated with sampling quality by the participants: when swabbing was poor, slides had a lower cell count, less than half of the total average. DAPI staining and overall slide quality were satisfactory. Nevertheless, cytoplasm staining is sometimes poorly defined in the microscope, which makes MN identification harder. It can be difficult to differentiate MNi from artifacts such as staining particles or debris.
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05/09/2018 10:57
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08/09/2020 7:10
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