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
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 counter
stain. Following acquisition, images from mBAND samples are delivered
to an image-analysis program written in Matlab. This algorithm
• 1) reads the gray-scale image (probe profile) for each fluorophore
• 2) applies digital conditioning
• 3) 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.