Cell-Free DNA Extraction of Vitreous and Aqueous Humor Specimens for Diagnosis and Monitoring of Vitreoretinal Lymphoma

With this research, we are attempting to use molecular testing of vitreous humor collected from the eye by vitrectomy to diagnose vitreoretinal lymphoma, or VRL, which is an aggressive disease whose symptoms overlap those of common inflammatory ocular conditions. Cytopathology, flow cytometry, and molecular testing are current methods used to diagnose VRL. Published sensitivities of non-molecular methods are 44 to 90%for cytopathology and 50 to 60%for flow cytometry.

Cytopathology, flow cytometry, and molecular testing with cellular DNA requires intact neoplastic cells. That poses a problem in vitreous humor specimens because the volume and cellularity of these specimens is low and cells can be degraded by apoptosis as well as specimen collection processing and storage. We found that cell-free DNA extracted from vitreous fluid specimens provides another source of DNA from molecular testing that does not require intact cells and that also shows evidence of VRL and can be used to diagnose this lymphoma.

Cell-free DNA extraction offers molecular testing without intact neoplastic cells. This allows maximizing the utility of vitreous specimens as intact cells can be reserved for cytopathology and flow cytometry, while utilizing the remaining supernatant fluid that would otherwise be discarded for molecular testing. To begin, dilute viscous vitreous fluid with three to five milliliters of PBS to facilitate pipetting.

Drain the dilute vitreous fluid received within a cassette bag into 50 milliliter tubes. For low volume aqueous humor, transfer the sample into a 1.5 milliliter tube, then rinse the original 500 microliter microcentrifuge tube with an equal volume of PBS. Add the rinse solution to the 1.5 milliliter tube.

Lastly, centrifuge the vitreous fluid at 3, 000 G for 15 minutes at room temperature. Then carefully pipette out the supernatant into a new tube without disturbing the cell pellet. To perform DNA extraction, first add an appropriate amount of cell lysis solution to the cell pellet obtained from the eye humor.

Pipette the solution up and down to mix it thoroughly. Once the cells are fully lysed, add protein precipitate solution to the make up 1/3 of the total volume. Now vortex and mix vigorously for 20 to 30 seconds.

Then spin it for three minutes at 3, 000 G at room temperature. Meanwhile, all transfer 300 microliters of isopropanol into a clean microcentrifuge tube. Once centrifugation is complete, carefully pipette the supernatant into the tube containing isopropanol.

Next, add 1.5 microliters of glycogen to the tube and invert the tube to mix thoroughly. Place the tube at minus 20 degrees Celsius in a freezer for an hour or overnight to facilitate DNA precipitation. After freezing, centrifuge the tube at five minutes at 3, 000 G at room temperature.

Then pipette out the isopropanol. To wash the pellet, add 0.5 milliliters of 70%ethanol. Invert the tube gently to ensure even washing.

Then spin the tube for one minute at 3, 000 G at room temperature. Discard the supernatant from the centrifuge tube. Now invert the tube on clean gauze to drain the solution and dry the pellet.

After a quick spin in the microcentrifuge, use a fine tip disposable pipette tip to pipette the remaining ethanol droplet. Next, add 45 microliters of DNA hydration solution to the dried DNA pellet. Then place the tube in a heating block set to 50 degrees Celsius.

DNA yields of both cellular and cell-free components of the eye fluids were similar. PCR analysis of the cellular and cell-free components of a vitreoretinal lymphoma associated-mutation was performed. A higher mutation rate was present in the cell-free component as demonstrated by a lowered cycle threshold.

To begin, add 70 microliters of conditioning buffer for every milliliter of the cell-free supernatant fraction of the eye humor. Vortex the clearing beads to mix thoroughly. Next, add the beads to the sample.

Then vortex the sample and bead mixtures. Centrifuge this mix at 3, 000 G for 15 minutes at room temperature. Without dislodging the pellet, extract the supernatant.

Then add an equal volume of urine pellet digestion buffer to the pellet. Pipette the solution up and down to resuspend the pellet. Now add proteinase K to the resuspended pellet mixture.

Incubate the mixture at 55 degrees Celsius for 30 minutes. Then add an equal volume of genomic lysis buffer to the digestion mixture. Vortex the solution thoroughly to mix it well.

Now place the commercially available spin column in a new collection tube. Add 200 microliters of urine DNA prep buffer to the spin column. Centrifuge at 16, 000 G for one minute at room temperature and discard the flowthrough.

Now pipette 700 microliters of urine DNA wash buffer to the column and centrifuge as demonstrated After discarding the flowthrough, transfer the spin column to a DNAse-RNAse-free microcentrifuge tube. Lastly, add the appropriate volume of DNA elution buffer directly to the column matrix and let it sit for three to five minutes at room temperature. Then centrifuge again to obtain cell-free DNA.

DNA yields of both cellular and cell-free components of the eye fluids were similar. PCR analysis of the cellular and cell-free components of a vitreoretinal lymphoma-associated mutation was performed. A higher mutation rate was present in the cell-free component as demonstrated by a lowered cycle threshold.