Antonella Bertucci

THE RABIT: A RAPID AUTOMATED BIODOSIMETRY TOOL FOR RADIOLOGICAL TRIAGE

In response to the recognized need for high throughput biodosimetry methods for use after large-scale radiological events, a logical approach is complete automation of standard biodosimetric assays that are currently performed manually. The authors describe progress to date on the RABIT (Rapid Automated BIodosimetry Tool), designed to score micronuclei or &ggr;-H2AX fluorescence in lymphocytes derived from a single drop of blood from a fingerstick. The RABIT system is designed to be completely automated, from the input of the capillary blood sample into the machine to the output of a dose estimate. Improvements in throughput are achieved through use of a single drop of blood, optimization of the biological protocols for in situ analysis in multi-well plates, implementation of robotic-plate and liquid handling, and new developments in high-speed imaging. Automating well-established bioassays represents a promising approach to high-throughput radiation biodosimetry, both because high throughputs can be achieved, but also because the time to deployment is potentially much shorter than for a new biological assay. Here the authors describe the development of each of the individual modules of the RABIT system and show preliminary data from key modules. System integration is ongoing, followed by calibration and validation.

Adapting the γ-H2AX Assay for Automated Processing in Human Lymphocytes. 1. Technological Aspects

Abstract The immunofluorescence-based detection of γ-H2AX is a reliable and sensitive method for quantitatively measuring DNA double-strand breaks (DSBs) in irradiated samples. Since H2AX phosphorylation is highly linear with radiation dose, this well-established biomarker is in current use in radiation biodosimetry. At the Center for High-Throughput Minimally Invasive Radiation Biodosimetry, we have developed a fully automated high-throughput system, the RABIT (Rapid Automated Biodosimetry Tool), that can be used to measure γ-H2AX yields from fingerstick-derived samples of blood. The RABIT workstation has been designed to fully automate the γ-H2AX immunocytochemical protocol, from the isolation of human blood lymphocytes in heparin-coated PVC capillaries to the immunolabeling of γ-H2AX protein and image acquisition to determine fluorescence yield. High throughput is achieved through the use of purpose-built robotics, lymphocyte handling in 96-well filter-bottomed plates, and high-speed imaging. The goal of the present study was to optimize and validate the performance of the RABIT system for the reproducible and quantitative detection of γ-H2AX total fluorescence in lymphocytes in a multiwell format. Validation of our biodosimetry platform was achieved by the linear detection of a dose-dependent increase in γ-H2AX fluorescence in peripheral blood samples irradiated ex vivo with γ rays over the range 0 to 8 Gy. This study demonstrates for the first time the optimization and use of our robotically based biodosimetry workstation to successfully quantify γ-H2AX total fluorescence in irradiated peripheral lymphocytes.

The RABiT: A Rapid Automated Biodosimetry Tool for radiological triage. II. Technological developments

Purpose: Over the past five years the Center for Minimally Invasive Radiation Biodosimetry at Columbia University has developed the Rapid Automated Biodosimetry Tool (RABiT), a completely automated, ultra-high throughput biodosimetry workstation. This paper describes recent upgrades and reliability testing of the RABiT. Materials and methods: The RABiT analyses fingerstick-derived blood samples to estimate past radiation exposure or to identify individuals exposed above or below a cut-off dose. Through automated robotics, lymphocytes are extracted from fingerstick blood samples into filter-bottomed multi-well plates. Depending on the time since exposure, the RABiT scores either micronuclei or phosphorylation of the histone H2AX, in an automated robotic system, using filter-bottomed multi-well plates. Following lymphocyte culturing, fixation and staining, the filter bottoms are removed from the multi-well plates and sealed prior to automated high-speed imaging. Image analysis is performed online using dedicated image processing hardware. Both the sealed filters and the images are archived. Results: We have developed a new robotic system for lymphocyte processing, making use of an upgraded laser power and parallel processing of four capillaries at once. This system has allowed acceleration of lymphocyte isolation, the main bottleneck of the RABiT operation, from 12 to 2 sec/sample. Reliability tests have been performed on all robotic subsystems. Conclusions: Parallel handling of multiple samples through the use of dedicated, purpose-built, robotics and high speed imaging allows analysis of up to 30,000 samples per day.

Chromosome Inter- and Intrachanges Detected by Arm-Specific DNA Probes in the Progeny of Human Lymphocytes Exposed to Energetic Heavy Ions

Abstract Pignalosa, D., Bertucci, A., Gialanella, G., Grossi, G., Manti, L., Pugliese, M., Scampoli, P. and Durante, M. Chromosome Inter- and Intrachanges Detected by Arm-Specific DNA Probes in the Progeny of Human Lymphocytes Exposed to Energetic Heavy Ions. Radiat. Res. 170, 458–466 (2008). We measured residual cytogenetic damage in the progeny of human peripheral blood lymphocytes exposed to 1 GeV/ nucleon iron ions or γ rays. Arm-specific DNA probes for chromosome 1 were used to detect aberrations as a function of dose in cells harvested 144 h after exposure. In addition, arm-specific mFISH was applied to samples exposed to a single dose of 2 Gy. These methods allowed the detection of interarm intrachanges (pericentric inversions) in addition to interchanges. The ratio of these types of aberrations (F ratio) has been proposed as a fingerprint of exposure to densely ionizing radiation. The fractions of aberrant cells in the progeny of cells exposed to iron ions were similar to those in the population exposed to γ rays, possibly because many rearrangements induced by heavy ions ultimately lead to cell death. Simple inter- and intrachanges were also similar, but more complex rearrangements were found in cells that survived after exposure to iron ions. We did not find a significant difference in the ratio of simple interchanges to simple intrachanges for the two radiation types. However, iron ions induced a much higher frequency of events involving both inter- and intrachanges. We conclude that these complex rearrangements represent a hallmark of exposure to heavy ions and may be responsible of the decrease of the F ratio with increasing LET reported in the literature in some in vitro and in vivo experiments.

Effect of Dose Rate on Residual γ-H2AX Levels and Frequency of Micronuclei in X-Irradiated Mouse Lymphocytes

The biological risks associated with low-dose-rate (LDR) radiation exposures are not yet well defined. To assess the risk related to DNA damage, we compared the yields of two established biodosimetry end points, γ-H2AX and micronuclei (MNi), in peripheral mouse blood lymphocytes after prolonged in vivo exposure to LDR X rays (0.31 cGy/min) vs. acute high-dose-rate (HDR) exposure (1.03 Gy/min). C57BL/6 mice were total-body irradiated with 320 kVP X rays with doses of 0, 1.1, 2.2 and 4.45 Gy. Residual levels of total γ-H2AX fluorescence in lymphocytes isolated 24 h after the start of irradiation were assessed using indirect immunofluorescence methods. The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was used to determine apoptotic cell frequency in lymphocytes sampled at 24 h. Curve fitting analysis suggested that the dose response for γ-H2AX yields after acute exposures could be described by a linear dependence. In contrast, a linear-quadratic dose-response shape was more appropriate for LDR exposure (perhaps reflecting differences in repair time after different LDR doses). Dose-rate sparing effects (P < 0.05) were observed at doses ≤2.2 Gy, such that the acute dose γ-H2AX and TUNEL-positive cell yields were significantly larger than the equivalent LDR yields. At the 4.45 Gy dose there was no difference in γ-H2AX expression between the two dose rates, whereas there was a two- to threefold increase in apoptosis in the LDR samples compared to the equivalent 4.45 Gy acute dose. Micronuclei yields were measured at 24 h and 7 days using the in vitro cytokinesis-blocked micronucleus (CBMN) assay. The results showed that MNi yields increased up to 2.2 Gy with no further increase at 4.45 Gy and with no detectable dose-rate effect across the dose range 24 h or 7 days post exposure. In conclusion, the γ-H2AX biomarker showed higher sensitivity to measure dose-rate effects after low-dose LDR X rays compared to MNi formation; however, confounding factors such as variable repair times post exposure, increased cell killing and cell cycle block likely contributed to the yields of MNi with accumulating doses of ionizing radiation.

Multicolour FISH analysis of ionising radiation induced micronucleus formation in human lymphocytes

Micronucleation of chromosomal DNA is an effective indicator of DNA damage and micronucleus (MN) analysis is a valuable tool for radiation biodosimetry studies. To gain a comprehensive knowledge of micronucleation process after ionising radiation (IR) exposure, whole genome-wide chromosome analysis is desirable. With this objective, multicolour fluorescence in situ hybridization (M-FISH) technique was utilised in the present study to characterise the chromosome content of spontaneous and IR-induced micronuclei in three human donors. M-FISH analysis revealed a radiation dose-dependant increase in the number of micronuclei with multi-chromosome material above 2 Gy and as many as 3-6 multicolour signals were detected in micronuclei after high γ-rays radiation doses (5-10 Gy). Involvement of each human chromosome material was more frequently detected in multicoloured micronuclei than in single-coloured micronuclei at high radiation doses (>2 Gy). Observation of dose-dependant increase in the MN frequency with multi-chromosome material may be due to misrepair of DNA double-strand breaks involving multiple chromosomes leading to asymmetric dicentric or ring chromosomes and acentric fragments. Chromosomes belonging to groups A (1, 2 and 3) and B (4 and 5) were frequently detected in 35-45% of the total micronuclei either as single entities or in combination with other chromosomes. Among the A and B groups, chromosome 1 material was consistently detected at high MN frequencies after radiation exposure in all the donors. Additionally, chromosomes 13 and 19 were more frequently observed in micronuclei than the expected frequency based on DNA content. Our whole genome approach utilising the M-FISH technique revealed that MN formation at high radiation doses might be complex involving multiple chromosome fragments. Understanding the fate and biological consequences of these multi-chromosome-containing micronuclei may provide key molecular insights for some aspects of IR-induced genomic instability and cancer development processes.

Biological dosimetry in the ENEIDE Mission on the International Space Station

Space radiation represents one of the major health hazards to crews of interplanetary missions. As the duration of space flight increases, according to International Space Station (ISS) and Mars mission programs, the risk associated with exposure to ionizing radiation also increases. Although physical dosimetry is routinely performed in manned space missions, it is generally accepted that direct measurement of biological endpoints (biological dosimetry) is necessary for a precise assessment of radiation risk in extraterrestrial activities. Chromosomal aberrations (CAs) in peripheral blood lymphocytes (PBLs) are particularly suitable to this purpose, as they can provide estimates of both equivalent radiation dose and risk. In this study, cytogenetic analysis was performed on PBL chromosomes of an Italian astronaut involved in two different 10-day missions on the ISS (Marco Polo, April 2002, and ENEIDE, May 2005). Blood samples were collected before and after flights. CAs were evaluated in either mitotic spreads or in prematurely condensed chromosomes (PCC) by Fluorescence in Situ Hybridization (FISH). In addition, blood samples were exposed to graded doses of X-rays in vitro before and after the flight and cytogenetic damage evaluated to investigate whether the space environment alters the sensitivity of human cells to ionizing radiation. The yield of baseline chromosomal aberrations was not modified following Marco Polo and ENEIDE mission. This is consistent with the low dose absorbed in these short-term space missions. Preliminary results from Marco Polo mission suggested a significant increase in intrinsic radiosensitivity of lymphocytes after landing compared to pre-flight and follow-up (6 months after landing) samples. However, this effect was not observed during the ENEIDE mission. The results suggest that intra-indi-vidual variations in radiosensitivity are significant, but they cannot be related to the space flight.