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

Abstract

We demonstrate the use of high-throughput biodosimetry platforms based on commercial high-throughput/high-content screening robotic systems. The cytokinesis-block micronucleus (CBMN) assay, using only 20 μl whole blood from a fingerstick, was implemented on a PerkinElmer cell::explorer and General Electric IN Cell Analyzer 2000. On average 500 binucleated cells per sample were detected by our FluorQuantMN software. A calibration curve was generated in the radiation dose range up to 5.0 Gy using the data from 8 donors and 48,083 binucleated cells in total. The study described here demonstrates that high-throughput radiation biodosimetry is practical using current commercial high-throughput/high-content screening robotic systems, which can be readily programmed to perform and analyze robotics-optimized cytogenetic assays. Application to other commercial high-throughput/high-content screening systems beyond the ones used in this study is clearly practical. This approach will allow much wider access to high-throughput biodosimetric screening for large-scale radiological incidents than is currently available.

FIG. 1

General scheme of the automated CBMN assay of peripheral human blood lymphocytes at Columbia Genome Center.

FIG. 2

Layout of the cell::explorer^™ at the Columbia Genome Center used as RABiT-II, showing the various subsystems: anthropomorphic robotic arm (A), the JANUS^® liquid-handling system (B), EL406^™ microplate washer (C), the FlexDrop dispenser (D), STX500 automated incubator (E), V-spin^™ automated centrifuge (F).

FIG. 3

Scheme of the automated peripheral human blood sample harvesting in cell::explorer after 70 h of cell culturing.

FIG. 4

Imaging and image analysis. Panel A: A set of 81 images captured by IN CELL Analyzer using 20× objective lens representing one well of 96-well imaging plate with fixed and DAPI-stained sample. Panel B: Cropped image from a well of 96-well imaging plate with a cell with 2 nuclei and micronucleus (yellow arrow) and panel C: the same cell (green circle) with 2 nuclei (magenta circles with green border) and micronucleus (green dot), as delineated by FluorQuantMN.

FIG. 5

Kit for blood sample collection developed for the implementation of the CBMN assay of peripheral human blood lymphocytes on commercial biotech robotic systems.

FIG. 6

Dose-response curves of the MN yields induced in human lymphocytes (from 8 donors) exposed to gamma rays produced by using automated RABiT-II approach. The error bars represent the upper and lower 95% confidence intervals.

FIG. 7

Fitted linear-quadratic dose-response calibration curve of the MN yields in human lymphocytes for pooled data from 8 healthy donor using our RABiT-II system.