Simultaneous transplantation of limbal stem cells may reduce recurrences of granular dystrophy after corneal transplantation: 2 long-term case reports.

To present 2 cases with long-term relapse-free intervals only after limbo-keratoplasty but not after conventional penetrating keratoplasty in granular dystrophy.Retrospective review of the patient charts and photographs taken during long-term follow-up of 2 cases with granular dystrophy, in which 1 eye received penetrating keratoplasty and the fellow eye received penetrating limbo-keratoplasty.In the first patient, 1 eye showed extensive recurrence of granular deposits 17 years after penetrating keratoplasty was performed while in the second eye two-thirds of the corneal transplant adjacent to the transplanted limbal area remained clear 12 years after the limbo-corneal transplant. In the second patient, 1 eye showed no signs of recurrence 5 years after limbo-keratoplasty, whereas a recurrence of granular corneal deposits occurred 18 months after surgery in the fellow eye.These cases show that the simultaneous transplantation of healthy donor limbus when performing penetrating keratoplasty may prolong recurrence in granular corneal dystrophy. Although we were unable to prove it on the molecular level, these clinical courses may support the hypothesis that a limbal transplant helps prevent a recurrence.

Corneal surface reconstruction using adult mesenchymal stem cells in experimental limbal stem cell deficiency in rabbits.

PURPOSE: To investigate the ability of mesenchymal stem cells (MSC) to transdifferentiate to corneal epithelial cells in experimental limbal stem cell deficiency in rabbits. METHODS: Total limbal stem cell deficiency was produced in 21 right eyes of 21 New Zealand rabbits; 6 eyes served as controls (group 1, G1). After removal of the conjunctival overgrowth, five eyes received amniotic membrane transplantation (AMT; G2). In four eyes, autologous limbal stem cell transplantation from the healthy eye was performed with AMT (G3). In another six eyes, enriched autologous MSC were injected under the amniotic membrane (AM) (G4). Within 280 days, corneoscleral discs were analysed for goblet cells, cytokeratin (CK) 3/12, connexin 43, ß(1) -integrin, CK 19, α-enolase, p63 and ATP-binding cassette transporter subtype G-2 (ABCG-2) distribution patterns. RESULTS: Cultivated MSC were positive for CK 3/12 and α-enolase, but negative for ABCG-2, p63 and connexin 43. On rabbit corneas, CK 3/12 was expressed in all corneal regions in all groups, but with significantly different intensities. Among all other parameters, expression levels of ABCG-2, ß(1) -integrin and connexin 43 were significantly different between the transplanted groups and the control group. After a mean follow-up time of 172 (47-280) days, goblet cells were rarely present in the central cornea (G1-4). CONCLUSION: CK 3/12 is not highly specific for differentiated corneal epithelium. Further, goblet cells are not a reliable marker for conjunctivalization in rabbits. Expression of ABCG-2, ß(1)-integrin and connexin 43 after mesenchymal stem cell transplantation may indicate their ability to maintain their stem cell character or to transdifferentiate to epithelial progenitor cells.

Operational post-keratopasty graft tolerance due to differential HLAMatchmaker matching.

PURPOSE: Penetrating keratoplasty can restore vision in corneal blindness. However, immunologic rejection threatens graft survival. Matching donors at swine leukocyte antigen (SLA)-class II convey allo-specific tolerance in a large animal kidney-transplantation model despite mismatches at SLA-class I. The same matching pattern seems to account for the blood transfusion effect in kidney transplantation. Relying on the molecular basis of HLAMatchmaker eplets, we assessed whether this finding would also apply to keratoplasty, and if it would enhance the benefit from matching human leukocyte antigen (HLA)-class I alone. METHODS: We retrospectively selected two independent cohorts comprising 586 and 975 penetrating keratoplasties. Our computations revealed a quantitative tolerogenicity factor analogous to the animal model. The number of mismatched HLA-class I eplets functioned as a factor for conventional histocompatibility. In the first cohort, we empirically determined the thresholds with the highest predictive power on graft rejection for both factors, and confirmed those thresholds in the second cohort. We applied Cox proportional hazards regression for these analyses. RESULTS: The thresholds with highest predictive power revealed 220 eplets(2) for the tolerance factor and 10 eplets for HLA-class I histocompatibility. The respective hazards ratios were 2.22 (p=0.04) versus 3.63 (p<0.01) in the first cohort and 2.09 (p<0.01) versus 1.51 (p=0.02) in the second, confirmatory cohort. The threshold factors proved to be additive in predicting immune reactions in both cohorts, (hazard ratios 2.66 in cohort 1 versus 1.70; p<0.01 in cohort 2). CONCLUSIONS: Operational tolerance may be inducible by balanced matching of HLA-class I and II HLAMatchmaker eplets. Furthermore, such tolerance is additive to histocompatibility at HLA-class I.

Long-term graft survival in penetrating keratoplasty: the biexponential model of chronic endothelial cell loss revisited.

AIM: To present a novel interpretation of the biexponential nature of chronic endothelial cell loss after penetrating keratoplasty (PK). We hypothesize that the fast component of endothelial cell loss reflects the endothelial cells of graft origin. The slow component might just reflect cell loss of the recipient endothelium. We investigate herein whether this hypothesis is in line with long-term survival in bullous keratopathy (BK: almost no endothelium in the recipient bed) and keratoconus (KK: recipient bed with plenty of endothelium). METHODS: We reviewed endothelial graft failures in PK for BK (n = 88) and KK (n = 87). Patients with immune reactions or a history of glaucoma were excluded. We built a statistical model to predict graft failures from biexponential endothelial cell loss and compared this data to the actual outcomes. RESULTS: After 15 years, the incidence of late endothelial failures was 8% in KK and 33% in BK. The 95% confidence intervals of the simulated outcomes corresponded completely to the actual outcomes during follow-up. CONCLUSIONS: Our novel interpretation of the biexponential model is in line with long-term data of PK for BK and KK. Our findings highlight the importance of the recipient bed endothelial reservoir on the long-term prognosis in PK.

Epithelial marker expression in Salzmann nodular degeneration shows characteristics of limbal transient amplifying cells and alludes to an involvement of the epithelium in its pathogenesis.

PURPOSE: To look at the epithelial nature of Salzmann nodular degeneration (SND) and its possible relation with the aetiology of the subepithelial collagen deposition. METHODS: Histological slides of 28 patients with SND were analysed for limbal and central corneal epithelial markers. Expression pattern of these markers in the basal layer of the epithelium was analysed and compared to the expression pattern in central corneal and limbal epithelium. Statistical analysis was performed by means of analysis of variance. RESULTS: Expression of the epithelial stem cell marker ABCG2 and p63 was low in SND. Expression of CK12, a marker for terminally differentiated epithelium, was low, as well. But, CK19 and Enolase-alpha expressions were significantly increased and resembled the expression pattern of transient amplifying cells (TAC) of the limbus. CONCLUSION: The epithelium in SND shows similar characteristics as TAC of the limbus and seems to be metabolically more active than the differentiated central corneal epithelium. This could be related to the deposition of subepithelial collagen fibrils seen in SND and points out a possible involvement of the corneal epithelium in the aetiology of Salzmann nodular degeneration.

Expression of p16 in conjunctival intraepithelial neoplasia does not correlate with HPV-infection.

The aim of our study was to identify the frequency of expression of p16(INK4a) (CDKN2A) and HPV (human papilloma virus) in different grades of conjunctival intraepithelial neoplasia (CIN).Twelve specimens including CIN I (2), II (3), III (5), and CIN with beginning invasion (2), as well as 15 control specimens, were stained with antibodies against p16(INK4a) and MIB1. The presence of HPV was examined by PCR.p16 as well as MIB1 were significantly elevated in CIN compared to control specimens (p<0.01) without correlation with the differentiation grade. Only two cases with CIN grade 3 contained HPV 16.As few control specimens also showed increased p16(INK4a) expression, p16(INK4a) seems not to be a very reliable marker for the exact determination of CIN. It could serve as an additional diagnostic tool besides the morphological characterization. Our study suggested that p16(INK4a) elevation is not associated with HPV infection.

Matching of the minor histocompatibility antigen HLA-A1/H-Y may improve prognosis in corneal transplantation.

BACKGROUND: Minor histocompatibility (H) antigens are peptides of allelic intracellular proteins that play an important role in human leukocyte antigen (HLA) matched transplantations. In an animal model of keratoplasty, minor H antigens have even been reported to exceed the immunogenicity of major H antigens (MHC). This investigation is to assess any benefit of matching the broadly expressed gender (H-Y) and HA-3 antigens in HLA-A1 donor positive human keratoplasty. METHODS: A total of 229 HLA-A1 donor positive keratoplasties were analyzed. A Cox proportional hazards model and Kaplan-Meier analysis were applied to estimate the effect of H-Y or HA-3 mismatches on rejection-free graft survival. RESULTS: Eighty-one cases were mismatched for H-Y (male donor to female recipient). A mean follow up of two years showed graft survival as high as 88% in the H-Y compatible group compared to only 77% in the H-Y mismatched group (P = 0.02). Eight out of 62 cases were mismatched for HA-3. No statistically significant influence of HA-3 matching on rejection-free graft survival was observed (85% vs. 73%, P=0.52). CONCLUSION: HLA-A1/H-Y matching and matching for other broadly expressed minor H antigens may further improve prognosis in keratoplasty.

An open prospective pilot study on the use of rapamycin after penetrating high-risk keratoplasty.

BACKGROUND: The purpose of this study was to prove efficacy and safety of systemic immunosuppression with rapamycin following penetrating high-risk keratoplasty. Rapamycin has shown its immunosuppressive potential in the rat keratoplasty model and is a component of several immunosuppressive protocols after solid organ transplantation. In this pilot study, we compared the efficacy and safety of rapamycin and mycophenolate mofetil (MMF). METHODS: Ten patients (group 1) undergoing high-risk keratoplasty were included in this study, receiving rapamycin as postoperative immunoprophylaxis. Rapamycin was administered orally once daily (blood trough level 4-10 ng/ml) for 6 months. Thereafter, it was tapered over 2 weeks. The control group (group 2) consisted of 24 patients who received 1000 mg MMF twice daily for 6 months. All of the patients received postoperative medication with fluocortolone 1 mg/kg/day (tapered over 3 weeks) and prednisolone acetate eyedrops 5 times per day (tapered over 5 months). RESULTS: Mean follow-up of all patients (n=34) was 739 days. No immune reaction was observed in groups 1 and 2 during the first 6 months under immunosuppression. Two immune reactions occurred in group 1, and five in group 2 within a 2-year follow-up. All of the immune reactions were reversible. The side effects observed in both groups were mostly reversible. CONCLUSIONS: Rapamycin and mycophenolate mofetil seem to be similarly efficacious in preventing immune reactions after high-risk keratoplasty, as long as they are administered. However, we observed a broad spectrum of side effects from rapamycin.

Expression of p63 in conjunctival intraepithelial neoplasia and squamous cell carcinoma.

BACKGROUND: p63 is a homologue of the tumour suppressor gene p53, which is expressed in human basal squamous epithelium. Some investigators maintain that p63 plays a role in the development of squamous epithelium and, despite its homology to p53, it is considered to act as an oncogene. This study investigated the expression of p63 in conjunctival intraepithelial neoplasia of different grades, and conjunctival squamous cell carcinoma and its correlation to the proliferation marker MIB-1. MATERIAL AND METHODS: Seventeen conjunctival specimens excised with the suspicion of either conjunctival intraepithelial neoplasia (CIN) or squamous cell carcinoma were diagnosed histologically as follows: 2 squamous cell carcinomas of the conjunctiva, 2 CIN grade I, 3 CIN grade II, 7 CIN grade III, 2 CIN with beginning invasion and 1 normal conjunctiva with no dysplasia. Sixteen microscopically-normal postmortem conjunctival specimens and normal conjunctiva, CIN and carcinoma specimens were stained immunohistochemically with antibodies against p63 and MIB-1. At least 500 cells per specimen were counted and the percentage of positively-stained cells of each antibody was calculated. RESULTS: A mean of 80% (57-89%) of the dysplastic cells from the CIN specimens stained positively with antibodies against p63, especially in the lower two-thirds of the epithelium, statistically significantly more compared with the normal specimens (9-55%, mean 36%, p<0.001). Nevertheless, we did not find a correlation between the percentage of p63-positive cells and the differentiation grade of the malignant specimens. MIB-1 positivity was shown by 0-1% of the cells in the normal postmortem controls, by 3-30% (mean 12%) of the cells in the basal and occasionally in the middle layer of the CIN specimens, and 16-61% (mean 23%) in the carcinoma specimens. CONCLUSION: In conjunctival intraepithelial neoplasia and squamous cell carcinoma of the conjunctiva, p63 is preferentially expressed in the immature dysplastic epithelial cells. Its staining does not correlate with MIB-1-expression, and therefore does not appear to be linked to cell proliferation.

Systemic mycophenolate mofetil avoids immune reactions in penetrating high-risk keratoplasty: preliminary results of an ongoing prospectively randomized multicentre study.

Recently, in a monocentre study mycophenolate mofetil (MMF) was demonstrated to be efficacious and safe in penetrating high-risk keratoplasty. Here, preliminary results of a randomized multicentre trial are presented. To date, 86 of 140 scheduled patients undergoing high-risk penetrating keratoplasty have already been randomized into the two study groups: 48 into the MMF group and 38 into the control group. All 86 patients received fluocortolon 1 mg/kg body weight/day, tapered within 3 weeks, and topical prednisolone acetate 1% tapered within 5 months. MMF was administered at a daily oral dose of 2 x 1000 mg for the first 6 postoperative months. Thereafter, MMF was tapered within 2 weeks. The proportion of grafts with immune reactions and side-effects were the main outcome measures. Within an average follow up of 9.2 +/- 6.6 months two patients developed reversible endothelial immune reactions in the MMF group after cessation of MMF application. In the control group, five reversible and three irreversible immune reactions were observed within an average follow up of 10.1 +/- 7.6 months. According to Kaplan and Meier analysis, the ratio of grafts without immune reactions was estimated 89% 1 year postoperatively in the MMF group, in contrast to only 67% in the control group (P = 0.03; log-rank test). Fifteen patients experienced side-effects, especially gastroenterotoxicity, tachycardia, arthralgia or systemic infections. All attributable side-effects were reversible. Systemic MMF may be an effective and safe immune modulating drug in the prophylaxis of immune reactions after penetrating high-risk keratoplasty.

Immunosuppression with cyclosporine A and mycophenolate mofetil after penetrating high-risk keratoplasty: a retrospective study.

BACKGROUND: Graft-prognosis after penetrating high-risk keratoplasty has improved considerably with the use of systemic immunosuppressive medications. In this retrospective investigation we analyzed the long-term results of 417 high-risk keratoplasties with systemic immunosuppression (cyclosporine A [CsA] or mycophenolate mofetil [MMF]). METHODS: A total of 417 high-risk keratoplasties with postoperative systemic immunosuppression were evaluated retrospectively: CsA has been given in 252 keratoplasties since 1987, aiming at blood trough levels of 120 to 150 ng/mL. Systemic MMF at a daily dose of 2 x 1 g was administered in 149 surgical procedures. After 16 high-risk keratoplasties, combined systemic immunosuppression with CsA and MMF was administered. Systemic immunosuppression was scheduled for 6 to 12 months. All patients received fluocortolone 1 mg/kg body weight per day, tapered over 3 weeks, and topical prednisolone acetate 1%, tapered over 5 months. RESULTS: Rejection-free graft survival after 1 year was 75% in the CsA group and 89% in the MMF group; 60% of the grafts in the CsA group and 72% of the grafts in the MMF group were rejection-free 3 years postoperatively (Kaplan-Meier log-rank test P=0.03). Clear graft survival after 1 and 3 years was 92% and 77% (CsA) and 96% and 87% (MMF), respectively. The MMF-treated patients showed fewer side effects than the CsA-treated patients. The side effects attributable to both drugs were reversible. CONCLUSIONS: We found a statistically significant, stronger effect of MMF compared with CsA in preventing immune reactions after high-risk keratoplasty, despite a shorter MMF administration compared with CsA. Both systemic immunosuppressants were shown to have comparable potency regarding clear graft survival and were well tolerated.

Topical FK506 as immunoprophylaxis after allogeneic penetrating normal-risk keratoplasty: a randomized clinical pilot study.

The purpose of this study was to evaluate for the first time the efficacy and safety of topical FK506 in patients undergoing penetrating normal-risk keratoplasty in a prospectively randomized clinical trial. Twenty patients were treated with FK506 0.06% three times per day for 6 months postoperatively. An additional 20 patients received five drops of prednisolone acetate 1% tapered within 6 months. All patients received 1 mg/kg bodyweight/day of systemic fluocortolon tapered within 3 weeks postoperatively. Clear graft survival, ratio of immune reactions and side effects were the main outcome measures. One year postoperatively all patients of the FK 506 group were free from immune reactions, in contrast to 84% in the steroid group (Kaplan-Meier values; P = 0.9 in the log rank test). None of the patients developed irreversible graft failure so far. In eight patients of the FK506 group premature withdrawal of the drug was deemed appropriate because of local side effects. FK506 might turn out to become an effective immunoprophylaxis in subjects undergoing penetrating normal-risk keratoplasty. Local discomfort should be further reduced.

Endothelial cell loss after autologous rotational keratoplasty.

PURPOSE: To investigate whether it may be possible to ascertain the influence of immunological factors on chronic endothelial cell loss by comparing chronic endothelial cell loss after autologous rotational penetrating keratoplasty and after homologous penetrating keratoplasty. METHODS: For six patients who had undergone autologous rotational penetrating keratoplasty the relative annual loss of endothelial cells was calculated by means of an exponential regression analysis. The findings were compared with those in a homogeneous historical control group (53 patients undergoing homologous penetrating keratoplasty for keratoconus). RESULTS: After autologous rotational keratoplasty relative annual loss of endothelial cells was 1.1%+/-2.6% (mean +/- standard deviation). Relative annual loss of endothelial cells in the control-group was 16.7%+/-20.8%. CONCLUSIONS: The results of the study lead to the assumption that immunological influences might be the main cause for chronic endothelial cell loss after homologous penetrating keratoplasty.

Penetrating limbo-keratoplasty for granular and lattice corneal dystrophy: survival of donor limbal stem cells and intermediate-term clinical results.

PURPOSE: To assess clinical follow-up data, and to identify donor epithelial cells after homologous penetrating central limbo-keratoplasty in patients with granular and lattice corneal dystrophies compared with patients who underwent conventional penetrating keratoplasty (PK). DESIGN: Mixed retrospective and prospective nonrandomized comparative case series. PARTICIPANTS AND CONTROLS: Twenty-six patients who underwent 33 limbo-keratoplasty procedures for granular or lattice corneal dystrophy since May 1995 and a historical control group of 24 patients who underwent 36 PK procedures between November 1986 and May 1995. METHODS: Postoperatively, all but 2 limbo-keratoplasty patients were treated with systemic immunosuppressants for 6 months. All patients received long-term topical immune prophylaxis with prednisolone-21-acetate 1% (2 drops per day). After obtaining informed consent, epithelial cells were harvested from 10 limbo-keratoplasty eyes of 8 patients with granular dystrophy and 7 limbo-keratoplasty eyes of 7 patients with lattice dystrophy. Conjunctival epithelium or buccal mucosal epithelium for recipient identification and corneal epithelial cells from 3 graft sites were harvested. Deep-frozen donor corneoscleral rims were analyzed to characterize donor features. Genetic analysis was performed by polymerase chain reaction of short tandem repeat (STR) loci. MAIN OUTCOME MEASURES: The ratio of dystrophy recurrences in the graft was clinically assessed. Donor features in epithelial cells were genetically established if at least 1 STR profile differed from that of the recipient. RESULTS: There were 5 recurrences in limbo-keratoplasty eyes with granular dystrophy and 2 recurrences in limbo-keratoplasty eyes with lattice dystrophy, compared with 15 and 6 recurrences in PK eyes, respectively. The differences between limbo-keratoplasty and PK were not statistically significant over time (log-rank test; P = 0.14 for granular dystrophy and P = 0.56 for lattice dystrophy; alpha error, 0.05). For genetic analysis, 12 of 17 samples were evaluated. Donor epithelial cells were detected in 5 of the 12 samples (42%). CONCLUSIONS: Limbo-keratoplasty tended to be associated with fewer recurrences of granular and lattice dystrophies. However, the difference was not yet statistically significant, probably due to the disappearance of the transplanted limbal stem cells over time. Genetic analysis confirmed the survival of transplanted limbal stem cells over several years in some limbo-keratoplasty eyes, which might correlate with less recurrence. Limbo-keratoplasty, therefore, is likely to represent a first step towards long-term recurrence-free survival of corneal grafts in patients with granular and lattice dystrophies.

Long-term results of allogeneic penetrating limbo-keratoplasty in total limbal stem cell deficiency.

OBJECTIVE: To determine the prognosis of allogeneic penetrating limbo-keratoplasty in patients with total limbal stem cell deficiency and to find out if donor limbal stem cells survive in the long run. DESIGN: Noncomparative prospective case series. PARTICIPANTS: Forty-eight patients with total limbal stem cell deficiency. INTERVENTION: Allogeneic penetrating limbo-keratoplasty. All patients received systemic cyclosporin A and/or mycophenolate mofetil in the postoperative course. Thirteen patients received grafts with 0 to 1 HLA mismatches in the HLA-A, HLA-B, and HLA-DR loci; 13 patients received grafts with 2 to 6 mismatches; and 22 patients received untyped grafts. MAIN OUTCOME MEASURES: Long-term clear graft survival and survival of donor limbal stem cells. RESULTS: Five years postoperatively, 65% of the grafts with 0 to 1 mismatches, 41% of the grafts with 2 to 6 mismatches, and 14% of the untyped grafts were clear centrally (estimation according to Kaplan-Meier log rank test, P = 0.03). Immunogenetic analysis of epithelial cells from the surface of the graft could be performed successfully in 7 of 9 patients and revealed donor DNA in the epithelium of 5 of these 7 patients up to 56 months postoperatively. CONCLUSIONS: Long-term survival of donor epithelium could be demonstrated immunogenetically in patients undergoing allogeneic penetrating limbo-keratoplasty. Human leukocyte antigen-matched grafts seem to deliver better results than untyped grafts. Progress with matching and immunosuppressive strategies may further improve current results.

Recurrent interface infiltration with hypopyon after astigmatic laser in situ keratomileusis on a penetrating corneal graft.

A 56-year-old woman was referred with recurrent interface infiltration and hypopyon after astigmatic laser in situ keratomileusis (LASIK) on a corneal graft. Pseudomonas aeruginosa was isolated as the causative pathogen. Penetrating keratoplasty had been performed 2 years before refractive surgery. After the antibiotic medication was tapered, 3 recurrences of interface infiltration with hypopyon were observed. Penetrating rekeratoplasty was deemed appropriate. Histological examination of the explanted corneal graft revealed anterior stromal neutrophil infiltration. This case illustrates that microbial pathogens brought underneath the flap by LASIK can persist months later despite antimicrobial treatment.

Beneficial effect of matching at the HLA-A and -B amino-acid triplet level on rejection-free clear graft survival in penetrating keratoplasty.

OBJECTIVE: The beneficial effect of human leukocyte antigen (HLA) matching on long-term prognosis in penetrating keratoplasty is now unequivocal but has to be weighed against the additional waiting period on an individual basis. HLAMatchmaker is a molecularly based algorithm for histocompatibility determination that can identify immunologically acceptable mismatches and thus potentially reduce time on the waiting list dramatically without negatively affecting prognosis. METHODS: The HLAMatchmaker algorithm (triplet-string matching) was applied on each of 545 normal-risk keratoplasties for which complete HLA type was known at split-level resolution. Two homogeneous groups were defined. Group I consisted of the 147 penetrating keratoplasties with up to 13 triplet-string mismatches (the typical upper limit of foreign in case of a single HLA-A or HLA-B allele mismatch) and was compared to the remaining 398 patients with more triplet mismatches (group II) using the Kaplan-Meier method and log-rank statistics. Analysis of clear graft survival on the basis of conventional HLA-A and HLA-B matching was performed as well. Reduction of time on the waiting list as compared to conventional HLA-A and HLA-B matching was predicted individually. RESULTS: Triplet-string matching yielded 85% rejection-free clear graft survival 3 years after penetrating keratoplasty in group I but only 76% in group II (P<0.05), whereas conventional HLA-A and HLA-B matching did not result in any statistically significant reduction of immune reactions because of lack of statistical power (P=0.08). Triplet-string matching (13 mismatches accepted) reduces median time on the waiting list by 80%. CONCLUSIONS: Triplet-string matching seems to improve mid- to long-term prognosis in penetrating keratoplasties while simultaneously reducing time on the waiting list in most cases. It should thus be considered for histocompatibility determination in penetrating keratoplasty.

HLA class I/II matching and chronic endothelial cell loss in penetrating normal risk keratoplasty.

OBJECTIVE: Chronic endothelial cell loss of the graft is very common after penetrating keratoplasty. The aetiology of this is unknown. Clinically, non-identifiable immune reactions have been suspected. Recently, we were able to demonstrate that proper human leucocyte antigen (HLA) matching is a suitable means to reduce classical immune reactions in normal risk keratoplasty patients. In this study, we therefore investigated whether HLA-matched grafts also experience less chronic endothelial cell loss. METHODS: A homogenous group of 223 normal risk keratoplasty patients was divided into six groups with different degrees of HLA matching (group 1 with unknown HLA data, group 2 with up to two mismatches, group 3 with three mismatches, group 4 with four mismatches, group 5 with five mismatches and group 6 with six mismatches on the HLA A, B, DR loci). All serological HLA A, B, C and all moleculargenetic HLA DRB, DRQB typings of donors and recipients were performed in a single laboratory accredited by the American Society for Histocompatibility and Immunogenetics. Only patients with at least three postoperative endothelial cell density values were included in the study. The slopes of the regression lines for each individual scatterplot of endothelial cell density values plotted against postoperative time (linear regression, lost cells/mm2/day), and after logarithmic transformation (exponential regression, annual relative loss of cells) were evaluated, respectively. RESULTS: There were no statistically significant differences between the six groups. CONCLUSION: Whereas proper HLA matching at present standards is already a suitable means to reduce identifiable immune reactions and to prolong graft survival even in normal risk keratoplasty patients, the same HLA matching procedures are not effective in reducing the extent of chronic endothelial cell loss. For several reasons this does not yet exclude, however, the possibility that the underlying cause of chronic endothelial cell loss is immunological.