The Cytokinesis-Blocked Micronucleus assay (CBMN) quantifies radiation-induced chromosome damage expressed as post-mitotic micronuclei, in once-divided cells. For the CBMN assay, we use the standard Fenech scoring criteria.
A new binarization routine was developed specifically for processing images of DAPI-stained lymphocytes, where staining intensity of micronuclei is often lower than that of the main nuclei. We have seen excellent agreement between the yield of automatically scored micronuclei and those scored manually from the same images, also up to 8 Gy, although the yield of Micronuclei saturates above 5 Gy, probably due to mitotic delay.
A flow of the Micronucleus assay as implemented in the RABiT is shown below:
The γ-H2AX assay scores DNA double-strand breaks by quantifying phosphorylation of the histone H2AX which co-localizes to them. For the γ-H2AX analysis, both images of Hoechst stained nuclei and the γ-H2AX foci, visualized with Alexa Fluor 555 are acquired. Rather than counting foci, as is the standard procedure at low doses, but which saturates around 2 Gy, the RABiT analysis scores the total fluorescence in each nucleus. This modification allows scoring doses up to 8 Gy or more. Although not useful for diagnosing acute exposures, due to the 24-48h persistence of the γ-H2AX signal, we have seen that this is a good marker for both probing radiation sensitivity and for quantifying chronic exposures (e.g. to ingested 137Cs).
We have commenced developing a RABiT-based biological assay to quantify dicentric chromosomes. The general concept is to use FISH telomeric probes and a counterstain to identify the end of each chromosome, and FISH centromeric probes to locate chromosome centromeres. Identified chromosomes containing two centromeric probes would thus be dicentrics. This “counting spots” based FISH dicentric assay will make the image analysis far more tractable, and thus automatable, than current morphometrically based image analysis approaches. An example is shown, with the telometric probes (red) the counter-stain (blue), and the centromere (green). The image analysis software will then find the centerline of the chromosome and from the number of green spots along that line, determine if the chromosome is dicentric.
In terms of automation, the flowchart below shows the assay as it will be performed in the RABiT.mBAND
A second new assay under development is multicolor chromosome banding (mBAND), using Fluorescence In Situ Hybridization (FISH). This is an established technique that yields a visual interpretation of sequence along a chromosome. By selectively painting regions along a specific chromosome (chromosome 5 in our implementation) with a combination of one or more paints. This assay is particularly sensitive to high LET radiations such as those from alpha emitters and neutron irradiations and hence is useful for assaying the upcoming neutron and 239Pu irradiations.
While sample-preparation protocol and imaging systems exist for the mBAND assay, we aim to automate the entire mBAND assay process from sample preparation to sample scoring, within the RABiT system. Our goal is to produce a high-throughput, automated mBAND assay to characterize chromosomal rearrangement as a function of radiation quality and/or quantity.
A computer-controlled imaging system has been built in-house to acquire multi-colored images of mBAND samples. The system has proven successful for acquiring, in sequence, the six images associated with the colors in an mBAND sample: DEAC, FITC, Spectrum Orange, Texas Red, Cy5, and DAPI counterstain. Following the acquisition, images from mBAND samples are delivered to an image-analysis program written in Matlab. This algorithm
- reads the gray-scale image (probe profile) for each fluorophore
- applies digital conditioning
- applies intensity thresholds to binarize each fluorophore signal.
Regions manifesting particular color combinations are portrayed in pseudo color and give rise to the 11 basic bands for chromosome 5.
Ongoing tests at the RARAF neutron facility have demonstrated that our imaging system and analysis algorithms are suitable for detecting translocations following neutron irradiation. These translocations are not seen with photon irradiations establishing the RABiT-BAND assay as a useful tool for quantifying the neutron component of the exposure.