Thomas John, MD Clinical Associate Professor Loyola University at Chicago Maywood, Illinois Ritika Patel Chicago Medical School

John T, Patel R: Confocal Microscopy of Graft Rejection in Descemetorhexis with Endokeratoplasty PURPOSE: To report the confocal microscopic findings in “real time” within the human cornea during graft rejection following DXEK surgery. PURPOSE: To report the confocal microscopic findings in “real time” within the human cornea during graft rejection following DXEK surgery. SETTING/VENUE: This study was performed in the office setting in Tinley Park, Illinois. SETTING/VENUE: This study was performed in the office setting in Tinley Park, Illinois. METHODS: Four eyes of 4 patients with active corneal endothelial graft rejection after DXEK were clinically evaluated, and serial images using ConfoScan 3 confocal microscope (Nidek Inc., Freemont, CA) from 3 of these patients were examined to evaluate the cornea from the endothelium to the epithelial surface and look for keratic precipitates (KPs), ‘active’ keratocytes, macrotracks (indicative of corneal edema) and changes in the morphology of endothelial cells in the grafted donor corneal disc. Data regarding number, morphology, and distribution of the KPs were recorded from 10 different fields. The numbers and morphology of active keratocytes versus inactive keratocytes in the stroma were recorded in 50 fields. The characteristics of macrotracks were also recorded from 50 fields. Automated morphometric analysis was done for cell density, pleiomorphism, and polymegathism of endothelial cells, and this was compared top that of normal endothelium. METHODS: Four eyes of 4 patients with active corneal endothelial graft rejection after DXEK were clinically evaluated, and serial images using ConfoScan 3 confocal microscope (Nidek Inc., Freemont, CA) from 3 of these patients were examined to evaluate the cornea from the endothelium to the epithelial surface and look for keratic precipitates (KPs), ‘active’ keratocytes, macrotracks (indicative of corneal edema) and changes in the morphology of endothelial cells in the grafted donor corneal disc. Data regarding number, morphology, and distribution of the KPs were recorded from 10 different fields. The numbers and morphology of active keratocytes versus inactive keratocytes in the stroma were recorded in 50 fields. The characteristics of macrotracks were also recorded from 50 fields. Automated morphometric analysis was done for cell density, pleiomorphism, and polymegathism of endothelial cells, and this was compared top that of normal endothelium. RESULTS: The average age of patients in this study was 75.5 years (range years). All patients were caucasian females. In 2 of the 4 patients the right eye was involved in the graft rejection. On confocal microscopy (CM), KPs were seen as highly reflective, dense, spherical or ovoid bodies that were adherent to the corneal endothelium. These KPs can be widely distributed or grouped together. The active keratocytes appear as large, hperfluorescent, fusiform or spindle shaped bodies with cytoplasmic processes extending within the stroma. These are much larger compared to the inactive keratocytes that appear dull and smaller. In addition, numerous macrotracks appear as curvilinear tracks that may be vertical, horizontal, or oblique within the field. The automated statistical analysis of the endothelial cells showed that although the total numbers of endothelial cells were within the normal limit, there was a difference in the mean cell size and shape of endothelial cells in those with corneal endothelial graft rejection as compared to normal endothelium without graft rejection. RESULTS: The average age of patients in this study was 75.5 years (range years). All patients were caucasian females. In 2 of the 4 patients the right eye was involved in the graft rejection. On confocal microscopy (CM), KPs were seen as highly reflective, dense, spherical or ovoid bodies that were adherent to the corneal endothelium. These KPs can be widely distributed or grouped together. The active keratocytes appear as large, hperfluorescent, fusiform or spindle shaped bodies with cytoplasmic processes extending within the stroma. These are much larger compared to the inactive keratocytes that appear dull and smaller. In addition, numerous macrotracks appear as curvilinear tracks that may be vertical, horizontal, or oblique within the field. The automated statistical analysis of the endothelial cells showed that although the total numbers of endothelial cells were within the normal limit, there was a difference in the mean cell size and shape of endothelial cells in those with corneal endothelial graft rejection as compared to normal endothelium without graft rejection. CONCLUSIONS: To our knowledge, this is the first study that shows the use of CM to evaluate in “real time” the changes in the corneal endothelium, stroma, and epithelium that occur as a result of corneal endothelial graft rejection after DXEK surgery. Distinct morphological features and distribution of KPs on the corneal endothelium were noted. The presence of ‘active’ keratocytes and corneal edema was also noted which may contribute to visual loss during corneal graft rejection. FINANCIAL DISCLOSURE: John, T = None FINANCIAL DISCLOSURE: John, T = None

John, T ASCRS 2008, Chicago, IL Case 1 Case 2 Case 4 S/P DXEK, Graft Rejection Case 3

Confocal Microscopy of Graft Rejection following DXEK Confocal Microscopy of Graft Rejection following DXEK Confocal microscopy of keratic precipitates. These appear as cells that are highly reflective, spherical (yellow box-arrow) or ovoid bodies (red box- arrow), that are adherent to the donor corneal endothelium. They can be scattered or can appear in clumps (blue curved-arrow). Anterior chamber is seen to the right (blue star). Confocal microscopy of keratocytes in acute DXEK-graft-rejection The active keratocytes appear as hyperfluorescent, fusiform shaped bodies (yellow arrows) compared to the inactive keratocytes (red star) that appear as dull, fusiform bodies.

Confocal Microscopy of Graft Rejection following DXEK Confocal Microscopy of Graft Rejection following DXEK Confocal microscopy of macrotracks (arrows) within the stroma following DXEK-graft- rejection. These appear as dark, curvilinear tracks within the stroma that can be horizontal, vertical or oblique. Confocal microscopy of DXEK, donor-recipient interface (curved arrow). Increased reflectivity is seen at the interface. John, T

Figure 12: Results of confocal microscopy of corneal graft rejection as detected months after DXEK surgery. (cpf = cells per field) Cell sides analysis (pleomorphism). Normal cell pleiomorphism is >59.6%, therefore results show that there is less variation in cell shapes after graft rejection.

Thomas John, MD Clinical Associate Professor Loyola University at Chicago Maywood, Illinois Ritika Patel Chicago Medical School