The RABiT: high-throughput technology for assessing global DSB repair

Abstract

At the Center for High-Throughput Minimally Invasive Radiation Biodosimetry we have developed a Rapid Automated Biodosimetry Tool (RABiT); this is a completely automated, ultra-high throughput robotically-based biodosimetry workstation designed for use following a large scale radiological event, to perform radiation biodosimetry measurements based on a fingerstick blood sample. High throughput is achieved through purpose built robotics, sample handling in filter-bottomed multi-well plates and innovations in high speed imaging and analysis. Currently, we are adapting the RABiT technologies for use in laboratory settings, for applications in epidemiological and clinical studies. Our overall goal is to extend the RABiT system to directly measure the kinetics of DNA repair proteins. The design of the kinetic/time dependent studies is based on repeated, automated sampling of lymphocytes from a central reservoir of cells housed in the RABiT incubator as a function of time after the irradiation challenge. In the present study, we have characterized the DNA repair kinetics of the following repair proteins: γ-H2AX, 53-BP1, ATM kinase, MDC1 at multiple times (0.5, 2, 4, 7, 24 hours) after irradiation with 4 Gy γ rays. In order to provide a consistent dose exposure at time zero, we have developed an automated capillary irradiator to introduce DNA DSBs into fingerstick-size blood samples within the RABiT. To demonstrate the scalability of the laboratory-based RABiT system, we have initiated a population study using γ-H2AX as a biomarker.

Towards precision prevention: Technologies for identifying healthy individuals with high risk of disease

“…The RABiT ( R apid A utomated Bi odosimetry T echnology) approach for (among other endpoints) high-throughput γ-H2AX measurements was initially developed as an ultra-high-throughput technology for biodosi-metric reconstruction of past radiation exposures, measuring micro-nucleus or γ-H2AX yields in a fingerstick of blood, with a throughput of ~30,000 samples per day [21]. This fully-automated high-throughput methodology has been adapted to assay global DRC through automated quantification of the time-dependent kinetics of the disappearance of γ-H2AX foci after a radiation challenge [22]. …”

“…A key aspect here is the use of a small, inexpensive automated capillary fingerstick irradiator [22]. The multi-well plates containing the cells are maintained in the RABiT incubator, and the post-irradiation, time-dependent γ-H2AX yield measurements are based on sequential automated samplings from these lymphocytes at five post-irradiation times from 0.5 to 24 h. A typical result from a study of 94 individuals is shown in Fig.…”

“…γ-H2AX yields and DNA repair kinetics as measured with the high-throughput fully-automated RABiT system [21,22], from a study of finger stick blood samples from 94 healthy individuals [23]. Experimental data and model fit (see [23]) pooled from 94 donors exposed ex vivo to 4 Gy gamma radiation, assayed at 0.5, 2, 4, 7 and 24 h post irradiation.…”

EPRBioDose 2013: EPR applications and biological dosimetry

“…An automated biodosimetry tool with high throughput was developed for use following large-scale radiological events on the basis of measurements of fingerstick blood samples. Attempts were presented to extend this tool to measure the kinetics of DNA repair proteins like c-H2AX (Turner et al 2014).…”

THE DECADE OF THE RABiT (2005–15)

“…An assay for quantifying DNA repair kinetics (28,29) was also developed (under NIEHS funding) by modifying the gamma-H2AX RABiT assay. In the modified assay, an irradiated sample is split and analyzed at multiple time points post irradiation, generating a curve as seen in Figure 4.…”