Fast Image Analysis for the Micronucleus Assay in a Fully Automated High-Throughput Biodosimetry System

Lyulko, Oleksandra V.; Garty, Guy; Randers-Pehrson, Gerhard; Turner, Helen; Szolc, Barbara; Brenner, David J.

doi:10.1667/rr13441.1

Cited by 31 publications

(39 citation statements)

References 49 publications

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“…For both blood volumes cultured for 48 h, while there is a quadratic dependence from 0-3 Gy, the rate of increase of MN per BNC begins to plateau toward 4 Gy. This result is not unexpected as a similar trend was noted at 5 Gy using standard culture time and volume conditions in recent work using the ISX-CBMN method (Rodrigues et al 2014b) and in other automated CBMN techniques (Lyulko et al 2014). After only 48 h of culture time, fewer cells would be expected to have passed through mitosis at higher doses as compared to 72 h of culture time with damaged cells cycling even more slowly than healthy cells.…”

Section: Discussionsupporting

confidence: 84%

“…The Rapid Automated Biodosimetry Tool (RABiT) developed at Columbia University can process 6,000 samples per day, and, while dose response calibration curves have been generated using the system, the number of BNCs scored at each dose point was an order of magnitude lower than the number of BNCs scored to generate similar curves using the ISX-CBMN method. In addition, the time to identify and score the MN and BNCs to generate one data point in each calibration curve was estimated at 22 min (Lyulko et al 2014). Therefore, using the standard CBMN culture time and volume conditions described in this work (72 h, 2 mL), the ISX-CBMN method is as rapid and accurate as any other currently available methods that use the CBMN assay to estimate blinded radiation doses between 0-4 Gy.…”

Section: Discussionmentioning

confidence: 99%

“…In addition to the 72 h culture time requirement, typical standard CBMN protocols involve culturing 500 mL to 2 mL of peripheral blood in a large volume of culture medium (5-20 mL) (McNamee et al 2009;IAEA 2011;Kacprzak et al 2013;Lyulko et al 2014). A reduction in the initial blood culture volume may eliminate the need for peripheral blood sampling by venipuncture, which is invasive and may not be possible depending on the nature of the emergency and the state of the patient.…”

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confidence: 99%

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Validation of the Cytokinesis-block Micronucleus Assay Using Imaging Flow Cytometry for High Throughput Radiation Biodosimetry

Rodrigues¹,

Beaton-Green²,

Wilkins³

2016

Health Physics

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The cytokinesis-block micronucleus assay can be employed in triage radiation biodosimetry to determine the dose of radiation to an exposed individual by quantifying the frequency of micronuclei in binucleated lymphocyte cells. Partially automated analysis of the assay has been applied to traditional microscope-based methods, and most recently, the assay has been adapted to an automated imaging flow cytometry method. This method is able to automatically score a larger number of binucleated cells than are typically scored by microscopy. Whole blood samples were irradiated, divided into 2 mL and 200 μL aliquots, cultured for 48 h and 72 h, and processed to generate calibration curves from 0-4 Gy. To validate the method for use in radiation biodosimetry, nine separate whole blood samples were then irradiated to known doses, blinded, and processed. Results indicate that dose estimations can be determined to within ±0.5 Gy of the delivered dose after only 48 h of culture time with an initial blood volume of 200 μL. By performing the cytokinesis-block micronucleus assay using imaging flow cytometry, a significant reduction in the culture time and volume requirements is possible, which greatly increases the applicability of the assay in high throughput triage radiation biodosimetry.

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Section: Discussionsupporting

confidence: 84%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Validation of the Cytokinesis-block Micronucleus Assay Using Imaging Flow Cytometry for High Throughput Radiation Biodosimetry

Rodrigues¹,

Beaton-Green²,

Wilkins³

2016

Health Physics

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“…Over the past years our group has developed such a platform, the RABiT (Rapid Automated Biodosimetry Tool). The RABiT completely automates three well-established biodosimetry assays, the CBMN [31, 32], the yield of phosphorylation of the histone H2AX (γ-H2AX assay) [33-35] and the dicentric assay [36]. The RABiT allows high throughput analysis of thousands of blood samples per day, providing a dose estimate of past radiation exposure that can be used to support clinical triage and treatment decisions [37].…”

Section: Introductionmentioning

confidence: 99%

In vitro RABiT measurement of dose rate effects on radiation induction of micronuclei in human peripheral blood lymphocytes

Bertucci

Smilenov

Turner

et al. 2016

Radiat Environ Biophys

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Developing new methods for radiation biodosimetry has been identified as a high priority need in case of a radiological accident or nuclear terrorist attacks. A large-scale radiological incident would result in an immediate critical need to assess the radiation doses received by thousands of individuals. Casualties will be exposed to different doses and dose-rates due to their geographical position and sheltering conditions, and dose-rate is one of the principal factors that determine the biological consequences of a given absorbed dose. In these scenarios high-throughput platforms are required to identify the biological dose in a large number of exposed individuals for clinical monitoring and medical treatment. The RABiT (Rapid Automated Biodosimetry Tool) is designed to be completely automated from the input of blood sample into the machine to the output of a dose estimate. The primary goal of this paper was to quantify the dose-rate effects for RABiT-measured micronuclei in vitro in human lymphocytes. Blood samples from healthy volunteers were exposed in vitro to different doses of X-rays to acute and protracted doses over a period up to 24 hours. The acute dose (ADR) was delivered at ∼1.03Gy/min and the low dose rate (LDR) exposure at ∼0.31Gy/min. The results showed that the yield of micronuclei decreases with decreasing dose-rate starting at 2Gy, whereas response was indistinguishable from that of acute exposure in the low dose region, up to 0.5Gy. The results showed a linear-quadratic dose-response relationship for the occurrence of micronuclei for the acute exposure and a linear dose-response relationship for the low dose-rate exposure.

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“…The RABiT was originally designed to automate two well-known, biodosimetry assays ( Figure 1): (a) the gamma-H2AX assay (12,13) , which quantifies DNA double-strand breaks by immunolabeling of phosphorylated histone H2AX and (b) the CBMN assay (14)(15)(16)(17) , which measures persistent chromosomal aberrations as post-mitotic micronuclei. As the gamma-H2AX assay is rather rapid (requiring only a few hours to provide a dose estimate, as compared with 72 h for the CBMN assay), it is the preferred assay.…”

Section: The Original Assaysmentioning

confidence: 99%

THE DECADE OF THE RABiT (2005–15)

Garty

Turner

Salerno

et al. 2016

Radiat Prot Dosimetry

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The RABiT (Rapid Automated Biodosimetry Tool) is a dedicated Robotic platform for the automation of cytogenetics-based biodosimetry assays. The RABiT was developed to fulfill the critical requirement for triage following a mass radiological or nuclear event. Starting from well-characterized and accepted assays we developed a custom robotic platform to automate them. We present here a brief historical overview of the RABiT program at Columbia University from its inception in 2005 until the RABiT was dismantled at the end of 2015. The main focus of this paper is to demonstrate how the biological assays drove development of the custom robotic systems and in turn new advances in commercial robotic platforms inspired small modifications in the assays to allow replacing customized robotics with 'off the shelf' systems. Currently, a second-generation, RABiT II, system at Columbia University, consisting of a PerkinElmer cell::explorer, was programmed to perform the RABiT assays and is undergoing testing and optimization studies.

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Fast Image Analysis for the Micronucleus Assay in a Fully Automated High-Throughput Biodosimetry System

Cited by 31 publications

References 49 publications

Validation of the Cytokinesis-block Micronucleus Assay Using Imaging Flow Cytometry for High Throughput Radiation Biodosimetry

Validation of the Cytokinesis-block Micronucleus Assay Using Imaging Flow Cytometry for High Throughput Radiation Biodosimetry

In vitro RABiT measurement of dose rate effects on radiation induction of micronuclei in human peripheral blood lymphocytes

THE DECADE OF THE RABiT (2005–15)

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