RABiT-II: Implementation of a High-Throughput Micronucleus Biodosimetry Assay on Commercial Biotech Robotic Systems

Repin, Mikhail; Pampou, Sergey; Karan, Charles; Brenner, David J.; Garty, Guy

doi:10.1667/rr011cc.1

Cited by 43 publications

(42 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The CBMN assay is one of the simplest cytogenetic biodosimetry assays to perform and score. It is therefore also the easiest to automate 53 . However, the conventional CBMN assay is geared towards uniform photon exposures and provides only the photon-equivalent total body dose, which may not be the most useful parameter in scenarios involving mixed neutron + photon or partial body exposures.…”

Section: Discussionmentioning

confidence: 99%

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

Shuryak

Turner

Perrier

et al. 2019

Preprint

Self Cite

View full text Add to dashboard Cite

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 R 2 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 R 2 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.biodosimetry approach in this scenario was to distinguish neutron + photon mixtures from pure photon exposures, and to quantify the neutron contribution.

show abstract

Section: Discussionmentioning

confidence: 99%

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

Shuryak

Turner

Perrier

et al. 2019

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…Moreover, our protocols for RABiT-II cell harvesting with the use of centrifuge [9,15,17] as well as described in this paper centrifuge-free RABiT-II approach were developed for keeping all reagents and plates at room temperature so that more workstations without automated heaters/coolers could be used for CBMN assay sample preparation. Thus, almost any ANSI/SLAC-format compatible robotic cell handling system meeting the minimum requirements of the centrifuge-free RABiT-II approach can be used for sample preparation with the use of the corresponding RABiT-II systems has the potential to increase the capacity for biodosimetry response during a large-scale radiological/nuclear event.…”

Section: Discussionmentioning

confidence: 99%

“…However, at larger scales, manual analysis of blood samples, even using a large cytogenetic network of labs [6,7] becomes infeasible and an increase in overall throughput via automation of triage radiation biodosimetry is needed. The CBMN assay is the most suitable biodosimetry method for automation [8,9].…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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

Repin

Pampou

Brenner

et al. 2019

Preprint

Self Cite

View full text Add to dashboard Cite

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 highthroughput 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.

show abstract

“…Whole chromosomes, as well as chromosome fragments, can be in micronuclei that lag behind at anaphase during nuclear division; the Mn form into small, rounded bodies surrounded by their own nuclear envelope and can persist up to 1 year after irradiation (3). The CBMN is a particularly useful biodosimetric tool for quantifying radiation-induced chromosomal damage for population triage due to the simplicity of Mn scoring and the availability of high-throughput fully automated systems (4)(5)(6)(7) (8). However, The accuracy of cytogenetically-based techniques such as CBMN for dose reconstruction decreases for doses higher than 5 Gy of purely photon irradiations (9)(10) (11).…”

Section: Introductionmentioning

confidence: 99%

Cytogenetically-based biodosimetry after high doses of radiation

Pujol-Canadell

Perrier²,

Cunha³

et al. 2019

Preprint

View full text Add to dashboard Cite

ABSTRACTDosimetry 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.

show abstract

RABiT-II: Implementation of a High-Throughput Micronucleus Biodosimetry Assay on Commercial Biotech Robotic Systems

Cited by 43 publications

References 27 publications

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

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

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

Cytogenetically-based biodosimetry after high doses of radiation

Contact Info

Product

Resources

About