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Epithelial DNA Fingerprint Analysis after Homologous Penetrating Central Limbo-keratoplasty in Granular and Lattice Dystrophy

1Spelsberg H., 1Reinhard T., 2Henke L., 3Berschick P., 1Sundmacher R.,
1Heinrich-Heine-Universität Düsseldorf, Universitäts-Augenklinik (Düsseldorf)
2Institut für Blutgruppenforschung (Köln)
3Landeskriminalamt NRW (Düsseldorf)

Purpose: For seven years now homologous penetrating central limbo-keratoplasty (LKP) has been performed in patients with granular or lattice corneal dystrophy in order to avoid recurrences. Up to now, survival of the transplanted limbal stem cells could clinically only be judged by lack or development of recurrences of the dystrophy in the graft. In this study epithelial DNA fingerprint analysis was performed to detect the persistance of donor epithelial cells. 
Method: After informed consent epithelial abrasion was performed in 11 LKP eyes from 8 patients with granular and 8 LKP eyes from 7 patients with lattice dystrophy. Conjunctival epithelium and corneal epithelial cells from the donor limbal tissue, the center of the graft and the graft margin opposite to the donor limbus were harvested and stored at –20°C in sterile BSS until analysis was done. Deep-frozen donor corneoscleral rims were analyzed to characterize donor features. DNA fingerprint analysis was performed by PCR of "short tandem repeat loci" (STR loci) and analysis of these products by Abi Prism 310 Genetic Analyzer. Donor features were proven, when at least one STR system showed donor features.
Results: Seven of 19 (37%) swabs were not evaluable at all. Nine swabs (47%) were excellent and 3 swabs (16%) were restrictedly evaluable. Donor epithelial cells could be clearly detected in five of these 12 swabs. Among the corresponding five eyes only one showed a recurrence of the dystrophy in the graft after a mean follow-up time of 37 months.
Conclusions: DNA fingerprint analysis allows for direct proof of donor derived epithelial cells. This confirms the survival of at least some transplanted limbal stem cells, which seems to be clinically correlated with lack of recurrences. Homologous penetrating central limbo-keratoplasty, therefore, is a first step for long-term recurrence-free survival of corneal grafts in patients with granular or lattice dystrophy. Further ways must be developed to increase the long-term survival of the transplanted limbal stem cells as many as possible.

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