P.I. David Brenner, Columbia University
Overview
The RABIT (Rapid Automated BIodosimetry Tool) is a completely automated, ultra-high throughput robotically-based biodosimetry workstation. It uses advanced, high-speed automated image analysis and robotics to examine tissue samples (e.g., a fingerstick of blood) quickly for quantitative indicators of radiation exposure (e.g., fragments of DNA; DNA repair complexes).
The basic system involves two well-characterized assays with all the processing being carried out in-situ in multi-well plates.
The γ-H2AX assay is a direct measure of the number DNA double strand breaks (DSB) which are present. It measures DSB by immune-staining the phosphorylated H2AX histone which localizes to them. γ-H2AX yields are quantified by integrating the fluorescent intensity within each nucleus. This assay gives a same day result, but requires that the blood samples are available within about 36 hours of irradiation.
The micronucleus assay quantifies radiation-induced chromosome damage expressed as post-mitotic micronuclei. An important advantage of the micronucleus assay is that the signal is comparatively stable for some months post exposure, with a biological half life of about 12 months, so the need for early acquisition of blood samples is removed. Due to the required culture time, analysis time for this assay is approximately 70 hours.
Samples collected within 36 hours of irradiation will be analyzed using the γ-H2AX assay. Following this time, the RABIT will be switched over to micronucleus mode and all subsequent samples analyzed using the micronucleus assay.
Sample collection
We anticipate multiple fingerstick collection sites following a radiological event, such as doctor’s offices, church halls, PoD (Point of Dispensing) sites, hospitals, etc. At these locations, field collection kits, consisting of lancets, bar-coded capillary tubes with matched personal data cards, alcohol wipes, and sample holders for 32 filled capillaries, will be used to collect the samples.
System Layout
The robotic biodosimetry workstation consists of four main modules: centrifuge, cell harvesting system, liquid/plate handling robot and dedicated image acquisition/processing system.
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A video of the RABIT in action: |
High-Throughput Imaging Systems for Biodosimetry
Current automated imaging systems have limited throughput, mostly due to their non-specificity. We have therefore built a dedicated high-throughput imaging system for performing the micronucleus assay exclusively, seeking creative solutions for rapid sample manipulation, automated focusing and image acquisition and analysis. The throughput of the imaging system currently under advanced stages of design and component testing is estimated at 5-6 minutes/96-well plate or 20,000-30,000 individual samples/day.
Sample manipulation
The motion of the sample is separated into two components: a slower coarse motion and a rapid fine motion. The coarse motion is performed by a high speed stage capable of few-g accelerations. This motion is used to move between adjacent samples (9 mm in 50 msec). The fine motion between fields of view within a single sample is performed, not by moving the sample but rather by steering light, using fast galvanometric mirrors as shown right. Typical transit times between adjacent fields of view of the microscope objective are ~1 msec.
Focusing
A major rate limiting step in automated imaging system is focusing. Typically, in order to get good image quality, microscope objective lenses have rather small depth of field and are sensitive to the roughness of the sample being imaged. Our solution is to place a weak cylindrical lens in the optics path. Using an appropriately selected lens, a fluorescent bead will be imaged as circular when in focus and as elliptical when out of focus (see movie below), the aspect ratio being proportional to the distance from focus. The left image shows the resulting ellipse, while the image right shows the object as imaged by regular optics. The object-lens distance can then be corrected in one step.
Image acquisition and processing
Analysis of the image is performed online, as it is grabbed. By using dichroic mirrors and two cameras, attached to the same frame grabber board we can simultaneously “see” the nucleus and cytoplasm (for the micronucleus assay) or the nucleus and the γ-H2AX fluorescence (for the γ-H2AX assay) and rapidly analyze the images. A third camera, preceded by a cylindrical lens is used for monitoring the focus. The figure above describes the lightpath process. The figures below describe the image analysis process for the two different types of assays that can be performed.
Chen, Y., Zhang, J., Wang, H., Garty, G., Xu, Y., Lyulko, O.V., Turner, H.C., Randers-Pehrson, G., Simaan, N., Yao, Y.L. and Brenner, D.J. Design and preliminary validation of a rapid automated biosodimetry Tool for high througput radiological triage. Proceedings of 2009 ASME/IEEE International Conference on Mechatronic and Embedded Systems and Applications (in press).
Garty, G., Chen, Y., Salerno, A., Turner, H., Zhang, J., Lyulko, O.V., Xu, Y., Wang, H., Simaan, H., Randers-Pehrson, G., Yao, Y.L., Amundson, S. A. and Brenner D.J. (2009). The RABIT: A Rapid Automated Biodosimetry Tool for radiological triage. Health Physics (in press).
Salerno, A., Zhang, J., Bhatla, A., Lyulko, O. V., Nie, J., Dutta, A., Garty, G., Simaan, N., Randers-Pehrson, G., Yao, Y. L. and Brenner, D. J.. "Design Considerations for a Minimally Invasive High-Throughput Automation System for Radiation Biodosimetry". In Proceedings of the Third Annual IEEE Conference on Automation Science and Engineering (CASE), Scottsdale, AZ, September 2007, pp. 846-852. IEEE Catalog Number: 07EX1754C. ISBN: 1-4244-1154-8. Library of Congress: 2007922962. [PDF]
Collaborating Institutions
Center for Radiological Research, Columbia University
Department of Mechanical Engineering, Columbia University