{"id":1611,"date":"2019-09-19T09:15:55","date_gmt":"2019-09-19T15:15:55","guid":{"rendered":"https:\/\/www.crpa-acrp-bulletin.ca\/?p=1611"},"modified":"2021-05-08T22:42:43","modified_gmt":"2021-05-09T04:42:43","slug":"the-adaptation-of-the-cytokinesis-block-micronucleus-assay-to-imaging-flow-cytometry-ladaptation-du-test-avec-micronoyau-du-bloc-de-cytocinese-vers-une-cytometrie-a-flux-dimages","status":"publish","type":"post","link":"https:\/\/www.crpa-acrp-bulletin.ca\/fr\/2019\/09\/19\/the-adaptation-of-the-cytokinesis-block-micronucleus-assay-to-imaging-flow-cytometry-ladaptation-du-test-avec-micronoyau-du-bloc-de-cytocinese-vers-une-cytometrie-a-flux-dimages\/","title":{"rendered":"The Adaptation of the Cytokinesis-Block Micronucleus Assay to Imaging Flow Cytometry \/ L\u2019adaptation du test avec micronoyau du bloc de cytocin\u00e8se, vers une cytom\u00e9trie \u00e0 flux d\u2019images"},"content":{"rendered":"
\n

R\u00e9sum\u00e9<\/h4>\n

Concours de communications \u00e9tudiantes Anthony J. Mackay<\/strong><\/p>\n

Chaque ann\u00e9e, le Comit\u00e9 des \u00e9tudiants et des jeunes professionnels organise, en combinaison avec le congr\u00e8s annuel de l\u2019ACRP, un concours de communications \u00e9crites pour les \u00e9tudiants.\u00a0Le concours est ouvert \u00e0 tous les \u00e9tudiants inscrits \u00e0 temps plein dans un programme reli\u00e9 aux sciences des rayonnements, dans un \u00e9tablissement d\u2019enseignement (coll\u00e8ge ou universit\u00e9) canadien. Les participants doivent soumettre un r\u00e9sum\u00e9 qui porte sur un sujet li\u00e9 \u00e0 un aspect du rayonnement.<\/p>\n

De toutes les participations re\u00e7ues, trois finalistes ont \u00e9t\u00e9 retenus et auront l\u2019occasion de pr\u00e9senter leurs travaux au congr\u00e8s lors d\u2019une s\u00e9ance pl\u00e9ni\u00e8re. Voici la gagnante de 2019.<\/p>\n

L\u2019adaptation du test avec micronoyau du bloc de cytocin\u00e8se, vers une cytom\u00e9trie \u00e0 flux d\u2019images<\/strong><\/p>\n

Apr\u00e8s un incident radiologique ou nucl\u00e9aire, une \u00e9valuation rapide de l\u2019exposition peut \u00e9ventuellement constituer le facteur d\u00e9cisif quand il s\u2019agit de sauver la vie d\u2019un patient. Ce groupe a adapt\u00e9 le micronoyau du bloc de cytocin\u00e8se \u00e0 une m\u00e9thode bas\u00e9e sur la cytom\u00e9trie \u00e0 flux d\u2019images, afin de cr\u00e9er un outil automatis\u00e9 de biodosim\u00e9trie \u00e0 rendement \u00e9lev\u00e9 pour am\u00e9liorer la m\u00e9thode de comptage traditionnelle (mais lente) bas\u00e9e sur la microscopie.<\/p>\n<\/div><\/div><\/div>\n

Anthony J. MacKay Student Paper Contest<\/h1>\n

Each year the Students and Young Professionals Committee organizes a student paper contest in conjunction with the CRPA annual conference. The contest is open to all students enrolled full-time in a Canadian college or university program related to radiation sciences. Students are invited to submit an abstract on a topic related to some aspect of radiation.<\/p>\n

From among the submissions, three finalists are selected and given the opportunity to present their work in a plenary session at the conference. Following is the 2019 winning paper.<\/p>\n

The Adaptation of the Cytokinesis-Block Micronucleus Assay to Imaging Flow Cytometry<\/h1>\n

by Riham Darwish
\nDepartment of Engineering, Carleton University, Health Canada, RPB<\/p>\n

Coauthors:<\/p>\n

J. J. Burtt, L. A. Beaton, N. J. Adam, S. Lachapelle, and R. C. Wilkins
\nConsumer and Clinical Radiation Protection Bureau, Healthy Environments and Consumer Safety Branch, Health Canada<\/p>\n

L. Yassin Kassab, Carleton University<\/p>\n

Q. Wang and H. Turner, Columbia University, New York<\/p>\n

Introduction<\/h2>\n

Following a radiological or nuclear event, a rapid dose assessment can inform medical providers as to the proper course of treatment for exposed individuals. This dose assessment tool can significantly decrease the required assessment period, ultimately being the deciding factor in saving the life of a patient.<\/p>\n

In order to achieve this goal, our group adapted the cytokinesis-block micronucleus (CBMN) to an imaging flow cytometry (IFC)-based method. These two techniques were paired to create an automated high-throughput biodosimetry tool to improve the traditional (time-consuming) scoring microscopy-based method.<\/p>\n

The CBMN assay is used to assess the frequency of chromosomal damage, specifically micronuclei (MN), in cells where cytokinesis has been blocked without inhibiting nuclear division. These cytokinesis-blocked cells thus produce binucleated cells (BNCs) rather than permitting the two daughter cells to separate. Damage inflicted by the radiation can be estimated by quantifying the frequency of MN per BNC derived from human peripheral blood lymphocytes.<\/p>\n

Method<\/h2>\n

Day 1<\/h3>\n

Blood samples from healthy donors who were diverse in age (25 to 60 years old), sex, and background were irradiated using the X-RAD 320 (precision X-ray) at an energy of 250 kVp and current of 12.5 mA in the dose range of 0\u201310 Gy. Following irradiation, blood samples were incubated for two hours at 37\u00b0C, and 5% CO2<\/sub> to allow for DNA repair. Then the blood was placed in culture media containing phytohemagglutinin (PHA), which stimulates the lymphocytes to proliferate.<\/p>\n

Day 2<\/h3>\n

After the blood samples had been incubated for 24 hours, cytochalasin B was added to block cytokinesis. Cytochalasin B stops the cells from dividing into two daughter cells.<\/p>\n

Day 3<\/h3>\n

We left the samples to incubate in order for the cells to proliferate and arrive at the cytokinesis stage. After the samples were incubated for 44 hours, the cells were fixed with FACS Lyse, which fixes the lymphocytes while lysing the red blood cells. Fixation of 22 samples requires two people and takes approximately two hours.<\/p>\n

Day 4<\/h3>\n

The cells were stained with a fluorescent DNA stain (DRAQ5), which is considered the most appropriate DNA stain in cell proliferation studies with the laser wavelength (642 nm) used on our cytometer. IFC analysis of the samples was performed on the IFC ImageStream\u00aeX<\/sup> Mk II using an autoplate that can hold up to 96 samples. This plate can be loaded directly onto the IFC ImageStream\u00aeX<\/sup> Mk II autosampler where analysis can be automatically performed for each dose point.<\/p>\n

Day 5<\/h3>\n

Data files were analyzed using the IDEA Data Analysis Software, which determined the total number of events, the BNCs, and the MN per BNC.<\/p>\n

Results<\/h2>\n

During this methods-development project, several experimental parameters were optimized to improve throughput:<\/p>\n