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An Automated Microscopic Scoring Method for the γ-H2AX Foci Assay in Human Peripheral Blood Lymphocytes
Published on: December 25, 2021
Adapting the γ-H2AX assay for automated processing in human lymphocytes. 1. Technological aspects
Helen C Turner1, David J Brenner, Youhua Chen
1Center for Radiological Research, Columbia University Medical Center, New York, New York 10032, USA. ht2231@columbia.edu
Radiation Research
|March 11, 2011
Summary
The Rapid Automated Biodosimetry Tool (RABIT) system automates DNA double-strand break (DSB) measurement using γ-H2AX in blood lymphocytes. This high-throughput method offers reproducible and quantitative radiation biodosimetry.
Area of Science:
- Radiation Biology
- Biotechnology
- Medical Diagnostics
Background:
- γ-H2AX detection quantifies DNA double-strand breaks (DSBs) and is a reliable biomarker for radiation biodosimetry due to its linear correlation with radiation dose.
- Current methods for DSB detection can be time-consuming and may not be suitable for high-throughput analysis.
Purpose of the Study:
- To optimize and validate the performance of the Rapid Automated Biodosimetry Tool (RABIT) system for reproducible and quantitative measurement of γ-H2AX fluorescence in lymphocytes.
- To assess the system's capability for high-throughput, minimally invasive radiation biodosimetry using fingerstick blood samples.
Main Methods:
- Development of a fully automated workstation (RABIT) to perform the entire γ-H2AX immunocytochemical protocol, including lymphocyte isolation, immunolabeling, and image acquisition.
- Utilizing robotics, 96-well filter-bottomed plates, and high-speed imaging for high-throughput processing of blood samples.
- Optimization and validation of the system using peripheral blood samples irradiated ex vivo with γ rays from 0 to 8 Gy.
Main Results:
- The RABIT system successfully automated the γ-H2AX immunocytochemical protocol for lymphocyte analysis.
- Demonstrated reproducible and quantitative detection of γ-H2AX total fluorescence in a multiwell format.
- Validated the biodosimetry platform by showing linear detection of a dose-dependent increase in γ-H2AX fluorescence in irradiated peripheral blood samples.
Conclusions:
- The RABIT system provides an optimized and validated platform for high-throughput, robotically based radiation biodosimetry.
- The system enables successful quantification of γ-H2AX total fluorescence in irradiated peripheral lymphocytes, offering a sensitive and reliable method for dose assessment.
- This automated approach advances minimally invasive radiation biodosimetry capabilities.
