An Automated Microscopic Scoring Method for the γ-H2AX Foci Assay in Human Peripheral Blood Lymphocytes

My name is Dr.Shankari Nair, and I'm a Postdoctoral Fellow at the Radiation Biophysics Division in the Nuclear Medicine Department at NRF, iThemba LABS. In this video, we will demonstrate the use of an automated Gamma H2AX foci assay on human peripheral blood lymphocytes for its use in batch symmetry applications. The radiation risks to personnel are strictly controlled.

Ionizing radiation directly affects the structure of DNA by inducing DNA breaks. Particularly double-strand breaks. One of the earliest steps in the DNA double-strand break recognition process is the phospohorilization of the histone variant, H2AX, and the recruitment of repair proteins.

This video will outline the use of microscopy to score the number of Gamma-H2AX foci ren-nucleus using an automated slide scanning system and analysis platform, integrating the fluorescent microscope. Lymphocytes are isolated from whole blood. Dilute whole blood with PBS in a one-to-one ratio, then gently lay the diluted blood onto one volume of density gradient medium.

Gradually layer the blood by holding the tube slanted at 45 degrees. Transfer the suspension to a centrifuge and spin at room temperature for 20 minutes at 900 G.After centrification four layers will form. Carefully transfer the cloudy layer containing the lymphocytes into a new sterile conical tube.

Wash the lymphocytes with PBS and count using a hemocytometer. Once the total number of lymphocytes are determined, dilute the lymphocyte and suspension to a concentration of approximately 800, 000 lymphocytes in a one milliliter of complete RPMI. The samples and are ready for our radiation.

The lymphocytes are irradiated with gamma rays from a cobalt 60 source, and thereafter incubated for one hour at 37 degrees Celsius in a humidified 5%carbon dioxide incubator. For every irradiation condition or tube, three slides are prepared. Place slide into the clip holder, followed by the filter card, and finally the funnel.

Secure slide clips and place into the cyto-centrifuge. Add 250 microliters of the cell suspension onto each funnel and spin for 30 G for five minutes. The use of the cyto-centrifuge is essential to get standardized conditions on the slides automation.

Once spun, remove the slides from the clip and then using the hydrophobic pen, draw a circle around the spot where the cells are. Now that the slides have been made, the cells need to be fixed to the slide so that the immunofluorescent staining can occur. The cells are fixed using a 3%paraformaldehyde, or PFA, and a PBS solution for 20 minutes.

After fixation wash slides in a Coplin jar with PBS for five minutes. Then, cover the cells with Triton X to allow permeabilization. The permeabilization step removes more cellular membrane lipids to allow antibodies to get inside the cell nucleus.

From here onwards, we use a blocking solution made of 1%BSA in PBS, to wash slides in order to reduce potential non-specific binding. After three wash steps of 10 minutes with 1%BSA and PBS, the slides are incubated at room temperature with one to 500 primary anti-gamma H2AX antibody for one hour in the humidifying chamber, easily accomplished by adding wet tissue paper at the base of the rectangular slide storage box. After incubation with a primary antibody, tip off the liquid and wash slides again in a 1%BSA solution.

Incubate slides with a one-to-one thousand diluted secondary donkey anti-mouse TRITC antibody for one hour in the humidifying chamber. Remove the antibody before performing three, 10-minute washes in PBS. Cover the Coplin jar in aluminum foil to prevent exposure to light for the duration of the wash steps.

Remove the slides and wipe off excess PBS outside the hydrophobic circle with dust-free tissue paper. Finally, add one to two drops of DAPI resulted in acquiesce mounting medium and gently cover the slides with the cover slip. Ensure that there are no trapped air bubbles and then store samples in a dark place at four degrees centigrade overnight.

Gently wipe slides with a 70%ethanol and place slides onto the automated scanning platform, or slide stage, attached to the zite axial images ZED-2 microscope. Select the classify set-up under the MetaCyte tab to edit the classifier. The selected classifier will be used by the system to scan and score the slides, and is based on a set of pre-defined parameters, which will determine how the system selects cells and scores foci.

The classifier can be optimized and trained based on a number of pre-classified image fields. Depending on the microscope magnification, the type of and antibodies that are used for the staining, a different classifier can be optimized. Therefore classifiers generally vary from laboratory to laboratory, but we will give an overview of the most basic steps taken to consideration when a classifier setup has to be adjusted.

Under the Capture'tab, one can find the filter that will be used, here DAPI and TRITC, with a maximum integration time of 1.04 seconds. The latter is based on the original classifier that was provided by the company and later adjusted for the antibody intensity and background gamma H2AX, or TRITC signal, in this specific cell type, namely lymphocytes. Under the Exposure'tab, the minimum integration time is defined.

The classifier will always remain within the minimum and maximum integration time, which prevents the over-or under-exposure of the slide. Under the Cell Selection'tab, the minimum and maximum object areas were defined based on the size and shape of the cells that are analyzed. For instance, the pre-determined concavity value and aspect ratio are specific for lymphocytes, which generally have round shapes.

Under the Features'tab, you can change the gallery and histogram view, as well as determine what the microscope needs to score. The system can score numerous items, depending on the features the user has selected. An example is the threshold of how the software scores individual objects.

Once a suitable classifier has been identified, select the setup in the sidebar and give each slide a unique name, followed by the selection of the appropriate classifier. Select Maximum Cell Count'and add the maximum number of cells required to be scanned. The search is terminated even if the selected search window has not yet been scanned completely, as soon as the max cell count is reached.

For bio-dissymmetry applications, automated scoring of 1000 cells per slide is sufficient, considering that three slides per blood sample are prepared. Click OK'to confirm the settings. To scan slides, select Search'in the sidebar.

If the setting Manual Search Window'was selected in the slide setup, a dialog will open a line for determination of the scan area. By using the 10-times objective the rectangular search area on the slide is selected interactively by fixing two corners of the search field by the left-click of the mouse. This can be confirmed with the OK'button.

Adjust a focus start position. The software will prompt a focus and center a reference nucleus using the 40 times objective for each slide and confirm settings with OK.The system will automatically move to all sides sequentially. If necessary, adjust Live Image Setup'and Integration Time'for this step.

Check all microscope settings. Make sure that the lever of the microscope tube is in a position that diverts all light to the camera and confirm the system prompt with OK.The system starts the grid auto-focus at the grid position closest to the reference field. It continues to focus field on the regular grid, moving in a meander towards the front and the back of the search window.

Scanning begins when the grid auto-focus is completed. The stage is moved in a meander pattern field after field to capture data. When a cell is detected, it's positioned and gallery image are stored and displayed in the screen and the cell count is updated.

The search is terminated if the whole search has been scanned, if the maximum cell count has been reached, or if the search has been canceled. When the scan is completed, right-click on the bar on the bottom and open a sample. Review the gallery, which consists of small images of detected cells.

In this window you can specify the cells stored in the gallery via criterion to be chosen from the dropdown menu. Cells are generally displayed in the order of which they have been detected. Click on Quality Histogram'or Feature Value Diagram'to give the distribution of the detected cells, as well as the means, and the standard deviation of foci scored.

Here we compare the zero gray versus 1.5 gray samples. This is typically a control slide, with a mean value of 0.124 foci per cell, where the cell hasn't been irradiated. In a bio-dissymmetry context, this would indicate that a person has not been exposed to a high-dose radiation.

In the graph, we can see that the majority of cells have almost zero foci per cell, and there is no side of normal distribution. When we compare this to a dose of 1.5 gray, it can clearly be seen that this is an irradiated sample. In a bio-dissymmetry context, this normal distribution is a clear sign that the sample has been exposed to radiation.

The automated scanning is sensitive enough to detect foci-induced at low doses. The results show a clear linear increase of the number of foci per cell with dose. The automated method of DNA double strand break detection is useful for fast and high throughput biological dyssymmetry applications.

Thank you for watching.