Can a digital slide scanner and viewing technique assist the visual scoring for the cytokinesis-block micronucleus cytome assay?

Abstract

The cytokinesis-block micronucleus cytome (CBMNcyt) assay is a comprehensive method to measure DNA damage, cytostasis and cytotoxicity caused by nutritional, radiation and chemical factors. A slide imaging technique has been identified as a new method to assist with the visual scoring of cells for the CBMNcyt assay. A NanoZoomer S60 Digital Pathology slide scanner was used to view WIL2-NS cells treated with hydrogen peroxide (H2O2) and measure CBMNcyt assay biomarkers using a high-definition desktop computer screen. The H2O2-treated WIL2-NS cells were also scored visually using a standard light microscope, and the two visual scoring methods were compared. Good agreement was found between the scoring methods for all DNA damage indices (micronuclei, nucleoplasmic bridges and nuclear buds) and nuclear division index with correlation R values ranging from 0.438 to 0.789, P < 0.05. Apoptotic and necrotic cell frequency was lower for the NanoZoomer scoring method, but necrotic frequency correlated well with the direct visual microscope method (R = 0.703, P < 0.0001). Considerable advantages of the NanoZoomer scoring method compared to direct visual microscopy includes reduced scoring time, improved ergonomics and a reduction in scorer fatigue. This study indicates that a digital slide scanning and viewing technique may assist with visual scoring for the CBMNcyt assay and provides similar results to conventional direct visual scoring.

Folic acid deficiency increases sensitivity to DNA damage by glucose and methylglyoxal

“…At days 7 and/or 14, an aliquot of cells was removed and subcultured to 3 × 10 5 cells/ml (in 0.5 ml of media) and treated with cytochalasin-B (cyt-B; 4.5 µg/ml) [ 23 ]. Cells were harvested 24 h later, and slide preparation and scoring of the CBMNcyt assay were performed as described previously [ 23 , 24 ] using a NanoZoomer S60 (Hammatsu Photonics).…”

Expression and localization of Sox10 during hair follicle morphogenesis and induced hair cycle

“…After washing in water, NanoZoomer S60 C13210 and NanoZoomer 2.0 HT digital pathology slide scanner (Hamamatsu Photonics, Hamamatsu City, Japan) were used in the present study to capture, view and score images. As previously reported 12 , each slide was scanned using a 20× or 40× objective lens of the NanoZoomer, and the slide code details, the scanning area and the number of focus points for each slide were determined by the user. Slide images were exported to an external storage device in Ndpi format and viewed using NanoZoomer Digital Pathology software (Hamamatsu Photonics, Hamamatsu City, Japan).…”

Methylglyoxal Impairs Sister Chromatid Separation in Lymphocytes

“…Briefly, following treatment with MGO for 24 or 48 h, cells were washed twice with Hanks balanced salt solution and resuspended in RPMI-1640 containing 4.5 µg/mL cytochalasin-b (cyt-b) for 24 h. After cytokinesis block, cells were harvested onto slides by cytocentrifugation [35]. Slide preparation and scoring of the CBMNcyt assay were performed as described previously using a NanoZoomer S60 (Hamamatsu Photonics, Shizuoka, Japan) [35,36]. Enlarged, misshapen, and multinucleated cells were prepared the same as for the CBMNcyt assay, except cytochalasin-b was not added following treatment of MGO.…”

“…After the fixative was removed, cells were resuspended in approximately 100 µL of fresh fixative and dropped onto microscope slides. Slides were air-dried and stained as described previously [36]. Metaphase spreads were scored microscopically using an Olympus CX43 at a 100 × objective lens.…”