Development of a high-throughput γ-H2AX assay based on imaging flow cytometry

• Published in: Radiation oncology (London, England) Vol. 14; no. 1; pp. 150 - 10
• Main Authors: Lee, Younghyun, Wang, Qi, Shuryak, Igor, Brenner, David J, Turner, Helen C
• Format: Journal Article
• Language: English
• Published: England BioMed Central 22-08-2019; BMC
• Subjects: Blood; Blood cells; Computer programs; Decay; Decay rate; Deoxyribonucleic acid; DNA; DNA damage; DNA Damage - radiation effects; DNA repair; DNA repair kinetics; Dose-Response Relationship, Radiation; Double-strand break repair; Exploration; Exposure; Flow cytometry; Flow Cytometry - methods; Fluorescence; Gamma Rays; High throughput; High-Throughput Screening Assays - methods; Histones - metabolism; Human lymphocytes; Humans; Imaging flow cytometry; Ionizing radiation; Irradiation; Kinetics; Lymphocytes; Lymphocytes - metabolism; Lymphocytes - radiation effects; Mathematical analysis; Matrix methods; Methodology; Peripheral blood; Precision medicine; Quantitation; Radiation; Radiation damage; Radiation dosage; Radiation effects; Radiation measurement; Radiation sensitivity; Radiation therapy; Radiation Tolerance - radiation effects; Radiosensitivity; Reaction kinetics; Repair; Robustness (mathematics); Software; Time dependence; High throughput; Radiation sensitivity; Ionizing radiation; DNA repair kinetics; Human lymphocytes; Imaging flow cytometry
• ISSN: 1748-717X; 1748-717X
• DOI: 10.1186/s13014-019-1344-7

Measurement of γ-H2AX foci levels in cells provides a sensitive and reliable method for quantitation of the radiation-induced DNA damage response. The objective of the present study was to develop a rapid, high-throughput γ-H2AX assay based on imaging flow cytometry (IFC) using the ImageStream®
Mk II (ISX) platform to evaluate DNA double strand break (DSB) repair kinetics in human peripheral blood cells after exposure to ionizing irradiation.
The γ-H2AX protocol was developed and optimized for small volumes (100 μL) of human blood in Matrix™ 96-tube format. Blood cell lymphocytes were identified and captured by ISX INSPIRE™ software and analyzed by Data Exploration and Analysis Software.
Dose- and time-dependent γ-H2AX levels corresponding to radiation exposure were measured at various time points over 24 h using the IFC system. γ-H2AX fluorescence intensity at 1 h after exposure, increased linearly with increasing radiation dose (R
 = 0.98) for the four human donors tested, whereas the dose response for the mean number of γ-H2AX foci/cell was not as robust (R
 = 0.81). Radiation-induced γ-H2AX levels rapidly increased within 30 min and reached a maximum by ~ 1 h, after which time there was fast decline by 6 h, followed by a much slower rate of disappearance up to 24 h. A mathematical approach for quantifying DNA repair kinetics using the rate of γ-H2AX decay (decay constant, K
), and yield of residual unrepaired breaks (F
) demonstrated differences in individual repair capacity between the healthy donors.
The results indicate that the IFC-based γ-H2AX protocol may provide a practical and high-throughput platform for measurements of individual global DNA DSB repair capacity which can facilitate precision medicine by predicting individual radiosensitivity and risk of developing adverse effects related to radiotherapy treatment.