Biology subjects

Garty, G.

Publications and source records attributed to Garty, G..

4 recordsLinked to original sources

bioRxiv2019

New High Throughput Approaches to Detect Partial-body and Neutron Exposures on an Individual Basis

Biodosimetry-based discrimination between homogeneous total-body photon exposure and complex irradiation scenarios (partial-body shielding and/or neutron + photon mixtures) can improve treatment decisions after mass-casualty radiation-related incidents. Our study objective was to use high-throughput biomarkers to: a) detect partial-body and/or neutron exposure on an individual basis, and b) estimate separately the photon and neutron doses in a mixed exposure. We developed a novel approach, where metrics related to the shapes of micronuclei distributions per binucleated cell in ex-vivo irradiated human lymphocytes (variance/mean, kurtosis, skewness, etc.) served as predictors in machine learning or parametric analyses of the following scenarios: (A) Homogeneous gamma-irradiation, mimicking total-body exposures, vs. mixtures of irradiated blood with unirradiated blood, mimicking partial-body exposures. (B) X rays vs. various neutron + photon mixtures. Classification of samples as homogeneously vs. heterogeneously irradiated (scenario A) achieved a receiver operating characteristic curve area (AUROC) of 0.931 (uncertainty range of 0.903-0.951), and R2 for actual vs. reconstructed mean dose was 0.87. Detection of samples with [≥]10% neutron contribution (scenario B) achieved AUROC of 0.916 (0.893-0.943), and R2 for reconstructing photon-equivalent dose was 0.77. These encouraging findings demonstrate a proof-of-principle for the proposed approach of analyzing micronuclei/cell distributions to detect clinically-relevant complex radiation exposure scenarios.

biophysics↗

bioRxiv2019

Cytogenetically-based biodosimetry after high doses of radiation

Dosimetry is an important tool for triage and treatment planning following any radiation exposure accident, and biological dosimetry, which estimates exposure dose using a biological parameter, is a practical means of determining the specific dose an individual receives. The cytokinesis-blocked micronucleus assay (CBMN) is an established biodosimetric tool to measure chromosomal damage in mitogen-stimulated human lymphocytes. The CBMN method is especially valuable for biodosimetry in triage situations thanks to simplicity in scoring and adaptability to high-throughput automated sample processing systems. While this technique produces dose-response data which fit very well to a linear-quadratic model for exposures to low linear energy transfer (LET) radiation and for doses up for 5 Gy, limitations to the accuracy of this method arise at larger doses. Resolution at higher doses is limited by the number of cells reaching mitosis. Whereas it would be expected that the yield of micronuclei increases with the dose, in many experiments it has been shown to actually decrease when normalized over the total number of cells. This variation from a monotonically increasing dose response poses a limitation for retrospective dose reconstruction. In this study we modified the standard CBMN assay to increase its resolution following exposures to higher doses of photons or a mixed neutron-photon beam. The assay is modified either through inhibitions of the G2/M and spindle checkpoints with the addition of caffeine and/or ZM447439 (an Aurora kinase inhibitor), respectively to the blood cultures at select times during the assay. Our results showed that caffeine addition improved assay performance for photon up to 10 Gy. This was achieved by extending the assay time from the typical 70 h to just 74 h. Compared to micronuclei yields without inhibitors, addition of caffeine and ZM447439 resulted in improved accuracy in the detection of micronuclei yields up to 10 Gy from photons and 4 Gy of mixed neutrons-photons. When the dose-effect curves were fitted to take into account the turnover phenomenon observed at higher doses, best fitting was achieved when the combination of both inhibitors was used. These techniques permit reliable dose reconstruction after high doses of radiation with a method that can be adapted to high-throughput automated sample processing systems.

cell biology↗

bioRxiv2019

The use of a centrifuge-free RABiT-II system for high-throughput micronucleus analysis

The cytokinesis-block micronucleus (CBMN) assay is considered as the most suitable biodosimetry method for automation. Previously, we automated this assay on a commercial robotic biotech high-throughput system (RABiT-II) adopting both a traditional and an accelerated micronucleus protocol, both using centrifugation steps for lymphocyte harvesting and washing, after whole blood culturing. Here we describe further development of our accelerated CBMN assay protocol for using on High Throughput/High Content Screening (HTS/HCS) robotic systems without a centrifuge. This opens the way for implementation of the CBMN assay on a wider range of commercial automated HTS/HCS systems and thus increases the potential capacity of dose estimates following a mass-casualty radiological event.

cell biology↗

bioRxiv2019

A new approach to automated CBMN scoring following high doses

In recent years we have automated the CBMN assay using microvolumes of blood, processed in multiwell plates. We have seen that at doses above 6 Gy the detected yield of micronuclei actually declines with dose, likely because of mitotic delay, preventing cells from forming micronuclei and also, when using one color imaging, resulting in many false binucleated cells, consisting of two randomly-adjacent nuclei. By using the inverse mitotic index (the ratio of mononuclear to binuclear cells) to adjust the micronucleus yield we were able to obtain a monotonic increasing dose response curve at doses of up to at least 10 Gy from the same samples which generated dose-response curve with a peak near 6 Gy, when scored using the traditional micronucleus yield.

cell biology↗