Automation of the Cytokinesis-Block Micronucleus Assay Using Imaging Flow Cytometry for High Throughput Radiation Biodosimetry

The cytokinesis-block micronucleus (CBMN) assay is employed in biodosimetry to determine the dose of radiation to exposed individuals from the frequency of micronuclei (MN) in binucleated lymphocyte cells (BNCs). The assay is traditionally performed using a microscope-based scoring procedure which can be labour-intensive, time consuming and subject to variability of interpretation between scorers. This thesis investigates the feasibility of adapting the CBMN assay to an imaging flow cytometry method using the ImageStreamX (ISX) imaging flow cytometer and hypothesises that dose estimations can be obtained with adequate sensitivity for triage radiation biodosimetry. A protocol to perform the CBMN assay on the ISX along with a data analysis method using the Image Data Exploration and Analysis Software (IDEAS®) were developed. Using irradiated whole blood samples it was shown that the ISX-CBMN method could automatically image and enumerate BNCs and MN. Additionally, between 0-4 Gy the rate of MN per BNC follows a linear quadratic dependence with dose, similar to what is observed when performing the assay using traditional protocols. The ISX-CBMN method was then expanded into a multi-parameter radiation biodosimetry tool. Irradiated whole blood samples were analyzed using an optimized version of the IDEAS® analysis template which incorporated automated scoring of mononuclear cells (MNCs). Dose response calibration curves for the rate of MN per BNC, percentage of MNCs and the replication index (RI) were generated. Results indicated that this multi-parameter method may be able to provide dose estimations for individuals who have received a radiation dose of higher than 4 Gy. The traditional CBMN assay is limited by the large blood culture volume (500 µL - 2 mL) and the 72 h culture time. It was posited that the ISX-CBMN method could overcome both of these requirements and results showed that dose estimations could be generated to within 0.5 Gy of the delivered dose after only 48 h of culture time with an initial blood volume of 200 µL. The adaptation of the CBMN assay to an imaging flow cytometry method greatly increases its applicability in high throughput triage radiation biodosimetry.