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EW CORNEA 104 April 2017 by Maxine Lipner EyeWorld Senior Contributing Writer retention, but at the potential risk of endothelial toxicity. While the lesser concentration (0.06%) showed no endothelial cell loss at 1, 3, or 5 minutes, with the higher concen- tration, at the 5-minute time period some cell loss was seen. "Most surgeons will place the graft into the stain right as the case starts," Dr. Majmudar said. "By the time we're ready to insert the graft, it might be 5 or 7 minutes, we're not really timing it." To insist that prac- titioners use the higher trypan blue concentration but rinse it out at, say, 3 minutes, would have been a burden, he stressed, adding that the fact was the higher concentration didn't really prolong the staining time significantly longer than the standard concentration. Investigators showed an average of 125 minutes of stain retention with the lower concentration left in the stain for 5 minutes, while at the higher concentration it was only 130 minutes, he reported. Granted, investigators didn't continue to examine the tissue at 6 hours, but 2 hours for the procedure is more than sufficient, Dr. Majmudar stressed. "I can do a routine DMEK procedure in about 20 minutes now," he said. "But even a novice surgeon would rarely take 2 hours to do the procedure." Overall, the study showed that trypan blue at 0.06% concentration centrations of the trypan blue and at different durations of application— how long can the corneal graft re- tain the stain, and furthermore, how long can it sit in the trypan blue before we see endothelial damage?" said Dr. Majmudar. Considering dye concentration After investigators evaluated various concentrations and different time points, they developed a recommen- dation for what the lowest concen- tration of trypan blue should be for staining the graft, Dr. Majmudar noted. "Anterior segment surgeons use trypan blue 0.06% often in staining the anterior capsule in cata- ract surgery, so that was an appropri- ate concentration for us to evaluate in DMEK," he said. "We found that staining the graft for time periods of 5–10 minutes will result in persistent staining for up to 2 hours." Most of the time, it will last longer than most DMEK surgeries will take, he pointed out, even in a surgeon's early cases. "We felt that this was an adequate amount of time for the stain to be present and so we recom- mended that the surgeons use the standard 0.06% Vision Blue [Dutch Ophthalmic Research Center, Zuid- land, the Netherlands] staining for 5 minutes, which would allow almost 2 hours of stain retention." The higher concentration of trypan blue (0.15%), typically used for posterior segment surgeries, did result in slightly longer stain "When trypan blue is used, not only can we visualize what the tissue is and where it is but we can actually see the orientation of it." The prob- lem is that the DMEK graft scrolls up, and it's important to make sure that the stromal side is the one that gets placed adjacent to the host cor- nea, he explained. "Otherwise if the endothelial side gets put up, then it's going to damage cells and the corneal graft is not going to work," he said. However, a new system where the eye bank places a micro S-stamp on the graft itself to help confirm orientation, was initiated before the study was published, Dr. Majmudar explained. While this made that aspect of the study moot, he added, you still need to stain the tissue with trypan blue in order to visualize and manipulate it. "It's really critical for novice DMEK surgeons as they're in the beginning of their learning curve, which can sometimes be fair- ly steep," he said. Another aspect of the study was the evaluation of how long the graft tissue retains trypan blue, and whether trypan blue is toxic to the endothelial cells, which no one had really examined before. Trypan blue retention in the graft is really important and if the procedure takes a longer period of time, as it often does early in the learning curve, the graft will become transparent again making the surgery more difficult. "We started looking at different con- How to remain in the pink with trypan blue S urgeons performing Descemet's membrane endothelial keratoplasty (DMEK) rely on trypan blue to visualize the corneal tissue, but just what is the best concentration of the dye to aid surgeons without harming tissue? In a recent study, published in the February 2017 issue of Cornea, inves- tigators considered what the optimal levels would be. 1 Studying visualization In general, visualization with trypan blue during DMEK is vital, according to Parag A. Majmudar, MD, associate professor of ophthal- mology, Rush University Medical Center, Chicago, who led the study. "We need to be able to manipulate and evaluate graft orientation, and if there is no staining at all, it is very difficult to identify the tissue because it's basically transparent," Dr. Majmudar said, adding that in cases of long-standing corneal edema, visualization is even further compromised because the cornea is so cloudy itself. When the study was launched initially, the major concern with DMEK was being able to identify the orientation of the graft. "Because the graft is clear we can't see it without trypan blue," Dr. Majmudar said. Eyeing DMEK tissue visualization DMEK graft immediately after staining for 5 minutes. Research highlight DMEK graft upon insertion into the eye. Note that the graft is in its normal "scrolled" orientation. Source (all): Parag A. Majmudar, MD continued on page 106

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