Biodosimetry estimation using the ratio of the longest:shortest length in the premature chromosome condensation (PCC) method applying autocapture and automatic image analysis

摘要

Peripheral blood samples from three healthy donors were obtained with informed consent according to the institutional ethical procedures of IRSN (Fontenay aux Roses, Paris). Samples were exposed to doses of 0, 1, 2, 5, 7.5, 10, 15 and 20 Gy (dose-rate 0.5 Gy/min) using a 60Co source of gamma radiation at the metrology laboratory of IRSN. The IAEA recommendations for cytogenetic dosimetry were followed during irradiations [1]. Briefly, blood samples were located inside a plexiglass holder that was submerged in a water bath heated to 37°C and placed in front of a 60Co gamma ray source during irradiation. The PCC chemically induced assay was conducted as described previously [8]. In all cases, 0.5 ml of peripheral whole blood was cultured for 48 h in 5 ml of RPMI 1640 medium containing L-glutamine, 20% fetal calf serum and 1% phytohaemaglutinin (PHA). Colcemid (0.05 μg/ml) was added 24 h after the beginning of the culture, and Calyculin A (50 nM) was added 1 h before the harvest. Cultured cells were treated with a hypotonic solution of KCl (0.075M) for 8 min at 37°C and fixed in three changes of fixative (methanol:acetic acid, 3:1 v/v). Finally, 30 μl of the resulting cell suspension was dropped onto slides, air-dried and stained with 4% Giemsa solution. Digitalized images at × 63 magnification were obtained automatically with a motorized AxioImager Z1 microscope (Carl Zeiss, Oberkochen, Germany) coupled to an AxioCam HRm camera (Carl Zeiss MicroImaging, GmbH, Jena, Germany) controlled by image analysis program Metafer 4 with MSearch and transmitted light (TL) mode. Images from the Metafer 4 gallery that had apparently complete sets of chromosomal pieces and no overlapped chromosomes were selected by an operator for analysis. A total of 25 images per subject were selected, since no statistically significant change was found in LR ratio by further increasing the number of images analysed. The images were saved as black and white images in TIF format. For image analysis, CellProfiler 2.0 (revision: 10997) software package for Windows was obtained from http://www.cellprofiler.org 19 March 2013, date last accessed [9]. The software uses a pipeline (available upon request from the authors) of modules designed to automatically identify, quantify and export the area–shape measurements of chromosome pieces with all PCC spreads analysed. For the CellProfiler pipeline setting, the semiautomatic (by manual drawing) and automatic measures were compared using Bland–Altman analysis, and a bias of ?0.3 pixels with a confidence interval from ?0.9 to 0.3 was obtained, which was considered negligible. The slope and the intercept of the dose–response linear regression were estimated with functional relationship by the maximum likelihood method considering in the model the mean value and the error calculated from the individual data of the three donors. The significance of the slope and the intercept were evaluated by t-test and the goodness-of-fit by chi-square test. The difference between donors was tested using the F test for comparing lines. The alpha level assumed for all tests was 0.05.