RABiT-II-DCA: A Fully-automated Dicentric Chromosome Assay in Multiwell Plates

Abstract

We developed a fully-automated dicentric chromosome assay (DCA) in multiwell plates. All operations, from sample loading to chromosome scoring, are performed, without human intervention, by the second-generation Rapid Automated Biodosimetry Tool II (RABiT-II) robotic system, a plate imager and custom software, FluorQuantDic. The system requires small volumes of blood (30 µl per individual) to determine radiation dose received as a result of a radiation accident or terrorist attack. To visualize dicentrics in multiwell plates, we implemented a non-classical protocol for centromere FISH staining at 37°C. The RABiT-II performs rapid analysis of chromosomes after extracting them from metaphase cells. With the use of multiwell plates, many samples can be screened at the same time. Thus, the RABiT-II DCA provides an advantage during triage when risk-based stratification and medical management are required for a large population exposed to unknown levels of ionizing radiation.

FIG. 1.

Comparison of the RABiT-II and the classical FISH staining with centromere probes. Panel A: Representative image of a dicentric stained with centromere probe at 37°C (without rehydration of samples in PBS, incubation in formaldehyde, pepsin treatment, dehydration in ethanol series and washes). Image captured at 20× magnification (BioTek Cytation Cell 1). Scale bar 2 μm. Panel B: Representative image of a dicentric after staining following classical FISH protocol at 80°C (including rehydration of samples in PBS, incubation in formaldehyde, pepsin treatment, dehydration in ethanol series and washes). Image captured at 63× magnification (Metafer 4 Master Station). Scale bar = 2 μm; dic = dicentric chromosome; cen = centromere.

FIG. 2.

Automated detection of dicentrics in the RABiT-II system. Panel A: Chromosomes released from mitotic cells (30 μl of whole blood), 0 Gy sample. Panel B: Output from the image analysis software FluorQuantDic version 3.3. Large blue objects are intact nuclei. White outlines correspond to identified monocentric chromosomes. Red outlines are objects which are either too big or too small to be scored as chromosomes. Green objects are too round to be chromosomes. Yellow objects are identified dicentric chromosomes. Inserts are identified dicentric (left) and monocentric (right) chromosomes. Panels from left to right: chromosome image (DAPI); the identified chromosome outline; centromere signal (FITC); identified centromeres; the mask for selecting which centromeres are associated with the chromosome; a merged DAPI/FITC image.

FIG. 3.

Assay development and quality control. Panel A: Caffeine allows G₂/M arrested lymphocytes to enter mitosis after severe DNA damage. Quantification data represents total number of mitotic figures scored in one well after 10 Gy irradiation without (286 mitotic cell spreads per well) or with (834 mitotic cell spreads per well) caffeine. Data and standard errors were calculated based on three independent experimental trials. Panel B: Frequency of second division metaphases in the RABiT-II DCA protocol after 52 h of culture. Images represent differentiation of sister chromatids by 33258 Hoechst fluorescence after different cycles of BrdU incorporation: first (bifiliar BrdU incorporation, on the left), second (unifiliar BrdU incorporation, in the middle), and third (one fourth BrdU incorporation, on the right) cell cycle metaphase spreads.

FIG. 4.

Uniform shapes of chromosomes after synchronization. Panel A: Common chromosomal morphologies frequently observed in the same well (from drop of blood from healthy volunteer, no irradiation, asynchronously cultured, 8 h incubation with colcemid). Images representing chromosome 1 were cropped and magnified from Supplementary Fig. S1 (http://dx.doi.org/10.1667/RR15266.1.S1). Panel B: FluorQuantDic classifier misrecognized the chromosome with opened arms as two chromosomes with closed arms. Panel C: Quantification data of mitotic figures before and after synchronization. Standard errors were calculated based on three independent experimental trials. Panel D: Proportion of mitotic cells with variable shapes of chromosomes (that can or cannot be detected using FluorQuantDic software) before and after synchronization. Panel E: Quantification results for distribution of chromosomal shapes before and after synchronization calculated for 1,000 prometaphase cells (0 Gy and 10 Gy irradiated samples). White and dashed populations represent cells with closed and open arms, respectively. Dark gray and black bars refer to undetectable or unpaired populations, respectively.

FIG. 5.

Schematic of the end-to-end automated RABiT-II DCA. The operating parameters are divided into several subprograms, which allows the user to perform various tasks including routine controls of the cell culture, synchronization, harvesting, fixation, staining and sample imaging.

FIG. 6.

Dose-response calibration curves for the RABiT-II and classical DCA. Panel A: Dicentric scoring strategy in the classical DCA. Representative example of a metaphase cell (10 Gy irradiated sample) suitable for analysis (complete set of centromeres, dicentrics are accompanied by acentric fragments, etc.). For each sample, analysis was performed in 50 metaphases using Isis software. Distribution of identified dicentrics (indicated by white arrows) was recorded; multicentrics are converted to the dicentric chromosome equivalents. Panel B: Dicentric scoring strategy in the RABiT-II DCA. Representative image (4 Gy sample) used for analysis. Scoring was performed automatically by the FluorQuantDic software (450 images per sample). A dicentric is indicated by the white arrow. Panel C: Preliminary dose-response curve for ¹³⁷Cs γ rays for classical DCA. The left axis shows the yield of DCs as a fraction of 50 cells scored. Panel D: Preliminary dose-response curve for ¹³⁷Cs γ rays performed using the RABiT-II system and scored without human intervention. The left axis shows the yield of dicentrics as a fraction of normal chromosomes.

FIG. 7.

The reliability of individual dose estimations by RABiT-II DCA and classical DCA. Four dose estimations of samples from two different donors (male and female, 29 and 30 years old, respectively) and the corresponding 95% CI were determined at each dose. The RABiT-II DCA and classical DCA calibration curves were used for dose-prediction calculations. Reconstructed doses are based on eight replicates for each donor; black line is an ideal reconstruction (reconstructed dose = physical dose) and dashed lines are ±20%.