Flow-sorting and Exome Sequencing of the Reed-Sternberg Cells of Classical Hodgkin Lymphoma

The overall goal of this procedure is to separate and concentrate viable Hodgkin Reed-Sternberg or HRS cells from inflammatory lymphocytic classical Hodgkin lymphoma or CHL tumors for the generation and analysis of high quality whole exome next generation sequencing data. This method can answer key questions in CHR biology, including identification of key driver mutations, immuno escape mutations, and also looking at genic diversity within the classification. This technique yields tumor derived DNA that is very high in quality and purity.

Facilitating the generation of next generation sequencing libraries without the need for a pre-amplification step. This technique may have important implications towards the diagnosis of Hodgkin lymphoma as it may help with a sub classification and prognostication. It may also influence a choice of therapy, as some alterations may indicate suscept-ivity or resistance to specific drugs.

Jonathan From first has this idea when he discovered how to visualize HRS cells by flow cytometry and perform the first sorting experiments with these cells. Before preparing the cells, mix the pre-titred antibodies in a dark glass vial. Then, add PBS and BSA to bring the antibody cocktail up to a 100 microliter volume.

And transfer the vial from liquid nitrogen into an ice bucket or dry ice. Next, rapidly thaw the cells of interest in a 37 degrees Celsius water bath. When only a small frozen portion remains, pour the cells into a 50 milliliter conical tube containing 45 milliliters of 37 degrees celsius thawing medium, and rinse the cryogenic vial two times with one milliliter of fresh thawing medium.

After pooling both rinses, allow the cells to digest at room temperature for 15 minutes. When the cells have re-equilibrated, collect them by centrifugation. Aspirate all but the last 200 microliters of the supernatant.

Re suspend the cells in the remaining thawing medium and allow the cells to rest for another two to three minutes. Then, incubate the cells in 100 microliters of the antibody cocktail for 15 minutes at room temperature, protected from light. At the end of the incubation, wash the cells in three milliliters of sorting medium, re-suspending the pellet in one milliliter of fresh sorting medium.

Then, filter the cells through a five milliliter flow tube top strainer. Rinsing the tube and the strainer with an additional one milliliter of sorting medium, and hold the cells on ice. Before beginning the cell sorting, in the flow cytometer software under the experiment menu, select new experiment and open the instrument status window.

In the parameters tab, delete any unused parameters. Then, reopen the experiment tab and select the compensation setup to create the compensation controls. In the browser window, click the plus sign to expand the compensation control specimen, and run the compensation control tubes without recording the data.

Adjust the detector voltages as necessary so that the positively stained bead populations for each flora chrome are between the 10, 000 and 100, 000 channel. And of the brightest in their primary detection channel. Record the voltages needed for each parameter for each tube.

Then, under the compensation control specimen, single left click to highlight the unstained control tube and load the unstained control tube onto the sample stage. In the acquisition control window, click load, and run the individual compensation controls into the detector voltage fields for all the parameters to manually enter the determined voltages. Click record to obtain the unstained control data.

Then run all of the remaining compensation controls, recording the data without changing any of the detector settings. When all of the controls have been analyzed, run a tube of sterile deionized water for five minutes to clear the instrument of any residual material before proceeding. To gate the cells for HRS sorting, load the experimental tube and record at least 100, 000 events while adjusting the flow rate to acquire 3, 000 to 4, 000 events per second.

Make sure you record enough events to visualize a population that can be as few as one in 1, 000 cells. To identify the somatic B and T cell controls, gate on the CD20, and CD5 positive cells respectively. When all of the gates have been set, sort the cells into pre chilled 15 milliliter conical tubes containing seven to eight milliliters of collection medium.

At the end of the sort, transfer the isolated cell populations into 1.5 milliliter micro centrifuge tubes. And pellet the cells by centrifugation. Then, wash the cells in one milliliter of fresh PBS and remove the supernatants from each tube, taking care not to disturb the tiny pellets.

After library amplification and point 8X bead cleanup, a normal light distribution of fragment sizes in the desired range should be observed. Libraries that exhibit deviations from this shape indicate the presence of a high or low molecular weight artifact, and ideally should not be sequenced. For example, and adapter dimer may sometimes carry over through the first pointed X bead cleanup, appearing as a sharp peak centered around 125 to 130 base pairs.

In this case, an additional pointed X bead clean up should be performed, followed by a repeat visualization of the size distribution to ensure the successful removal of the dimer. After multiplexing sequencing of four samples per lane, a median depth of coverage of 50 to 100 X across the target is achievable. Although copy number plots generated from a one nanogram library may results in spurious copy number gains and losses.

Approximately 70%of cases of classical Hodgkin lymphoma carry B2M and 820 mutations, and or deletions that appear to be clonal, and that can be used to estimate the relative tumor content. If T cells are used as the somatic controls, HRS sequencing will demonstrate significant copy number gains in the positions corresponding to the T cell receptor alpha, delta, and beta positions. As well as significant losses in the B cell receptor heavy, and kappa light chain positions with an overall mutational burden of 100 to 400 somatic mutations per exome case.

Once mastered, this technique including the cell sorting and the sequencing library construction, can be completed in three to four days if it is performed properly. Following this procedure, we can perform larger prospective studies and other methods, like epigenetics, proteomics, and metabalomics to gain more knowledge about Hodgkin lymphoma and progress towards more specific personalized therapies. After its development the method allowed us to study HRS cells with high purity in relatively large numbers.

After watching this video, you should have a good understanding of how to isolate HRS cells and to initiate their genomic analysis.