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 fingerstick-derived 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 heparin-coated 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.

FIG. 1

RABIT input stage. Four centrifuge buckets each holding 96 capillaries are manually loaded into the input stage of the RABIT workstation (panel A); the SCARA robot automates the transfer of each bucket in the centrifuge (panel B).

FIG. 2

Cell harvesting module. After centrifugation, the gripper arm of the SCARA robot positions the capillary tube containing the separated blood cells (panel A; arrow marks the location of the isolated lymphocyte band) in front of the UV laser (panels B, C). The laser cuts the capillary tube between the isolated lymphocyte band and RBC pellet (panel D), and the upper part of the capillary tube containing the lymphocyte band is pressure released into the microwell (panel E).

FIG. 3

Fluorescence of AF555 as a function of nuclear area at 0 and 1 Gy. The symbols correspond to fluorescence values F_i for individual unirradiated nuclei (closed symbols; values for 50 nuclei are shown) and nuclei irradiated with 1 Gy (open symbols; values for 16 nuclei are shown). For the unirradiated lymphocytes, the measured fluorescence is seen to depend linearly on nuclear area.

FIG. 4

RABIT optimization tests. The RABIT system was optimized for positive filtration pressure and the position of the laser to cut the capillary tubes to release the lymphocyte band into the filter plates. The panels show representative images of the results as determined by the appearance of Hoechst-labeled nuclei observed on the multiwell plate polycarbonate membranes. The lymphocytes were classified as either unacceptable (panel A), suboptimal (panel B) or optimal quality (panel C). Arrows show lymphocytes of normal rounded size and shape and arrowheads show the presence of distorted, irregular shaped or fragmented cells. Images were captured with a 20× objective lens.

FIG. 5

γ-H2AX expression in human lymphocytes. Representative γ-H2AX staining in isolated lymphocytes irradiated with 0 to 8 Gy, visualized with Alexa Fluor 555 for cells fixed at 30 min postirradiation. The green outlines denote the boundaries of the nuclei.

FIG. 6

Dose response for γ-H2AX analyzed 30 min after irradiation. Panel A shows a plot of the average total γ-H2AX fluorescence for the four healthy donors. Curve-fitting analysis showed that the induction of total γ-H2AX fluorescence was linear with increasing γ-ray dose up to 8 Gy. Panel B shows independent γ-H2AX fluorescence for the individual donors. Error bars are ±SEM.