Adapting the γ-H2AX Assay for Automated Processing in Human Lymphocytes. 1. Technological Aspects

Abstract

The immunofluorescence-based detection of γ-H2AX is a reliable and sensitive method for quantitatively measuring DNA double-strand breaks (DSBs) in irradiated samples. Since H2AX phosphorylation is highly linear with radiation dose, this well-established biomarker is in current use in radiation biodosimetry. At the Center for High-Throughput Minimally Invasive Radiation Biodosimetry, we have developed a fully automated high-throughput system, the RABIT (Rapid Automated Biodosimetry Tool), that can be used to measure γ-H2AX yields from fingerstickderived samples of blood. The RABIT workstation has been designed to fully automate the γ-H2AX immunocytochemical protocol, from the isolation of human blood lymphocytes in heparincoated PVC capillaries to the immunolabeling of γ-H2AX protein and image acquisition to determine fluorescence yield. High throughput is achieved through the use of purpose-built robotics, lymphocyte handling in 96-well filter-bottomed plates, and high-speed imaging. The goal of the present study was to optimize and validate the performance of the RABIT system for the reproducible and quantitative detection of γ-H2AX total fluorescence in lymphocytes in a multiwell format. Validation of our biodosimetry platform was achieved by the linear detection of a dose-dependent increase in γ-H2AX fluorescence in peripheral blood samples irradiated ex vivo with γ rays over the range 0 to 8 Gy. This study demonstrates for the first time the optimization and use of our robotically based biodosimetry workstation to successfully quantify γ-H2AX total fluorescence in irradiated peripheral lymphocytes.

Advances in a framework to compare bio-dosimetry methods for triage in large-scale radiation events

“…Time to emesis (EMESIS) 10 min to 4 h (27 -31) accurate recall: 48 h to .10 d ,5 min Micronucleus (CBMN) 0 to 1 d (24,32) Year (33) 4 to 6 d Gamma (H2AX) 3 to 30 min (7,34,35) 1 to 48 h (7,11,34,35) 1 to 2 d EPR in vivo tooth (EPR) 0 (17,36) Lifetime (17,37,38) ,10 min Gene expression 24 h (20) Variable by type (20) 2.5 to 17 d a W3 ¼ total processing times across five process steps; Table 2 presents references and details about the five steps.…”

“…the rapid automated bio-dosimetry tool for radiological triage (RABiT) (7,11,19) for gamma-H2AX (H2AX) or cytokinesis-block micronuclei analysis (CBMN) or computer-assisted readings for di-centric chromosome assay (DCA) (14). and for CBMN (45) .…”

Deoxyribonucleic acid damage-associated biomarkers of ionising radiation: current status and future relevance for radiology and radiotherapy

“…As a biodosimetry technique, it is useful immediately after exposure, as foci levels peak at ,1 h but then decline within days [34]. Developments have been made to increase speed and throughput of results by measuring g-H2AX intensity instead of scoring the number of foci [35], but this, as with most automated techniques, results in lower sensitivity. As this technique is mainly useful within hours of exposure, a recent study has aimed at developing a portable fluorescence spectrometer for use in triage of exposed individuals [36].…”

Suitability of the γ-H2AX Assay for Human Radiation Biodosimetry

“…analysis γ-H2AX total fluorescence measurements were determined in lymphocyte nuclei from blood samples collected from four healthy donors, irradiated (ex vivo) with a range of γ-ray doses between 0 and 8 Gy (Turner, 2011). Dose response for γ-H2AX was analyzed 30 min after irradiation.…”

“…Recently, a dual method has been developed to determine fluorescence yield using high-speed microscope imaging analysis. This workstation has been designed to fully automate the γ-H2AX immunocytochemical protocol, from the isolation of human blood lymphocytes to the image acquisition step (Turner, 2011). The translation of γ-H2AX analysis into a reliable dosimetry device nonetheless requires both further validation and better automated methods in microscopy-based scoring.…”