Fully automatic evaluation of the corneal endothelium from in vivo confocal microscopy.

BACKGROUND: Manual and semi-automatic analyses of images, acquired in vivo by confocal microscopy, are often used to determine the quality of corneal endothelium in the human eye. These procedures are highly time consuming. Here, we present two fully automatic methods to analyze and quantify corneal endothelium imaged by in vivo white light slit-scanning confocal microscopy. METHODS: In the first approach, endothelial cell density is estimated with the help of spatial frequency analysis. We evaluate published methods, and propose a new, parameter-free method. In the second approach, based on the stochastic watershed, cells are automatically segmented and the result is used to estimate cell density, polymegathism (cell size variability) and pleomorphism (cell shape variation). We show how to determine optimal values for the three parameters of this algorithm, and compare its results to a semi-automatic delineation by a trained observer. RESULTS: The frequency analysis method proposed here is more precise than any published method. The segmentation method outperforms the fully automatic method in the NAVIS software (Nidek Technologies Srl, Padova, Italy), which significantly overestimates the number of cells for cell densities below approximately 1200 mm(-2), as well as previously published methods. CONCLUSIONS: The methods presented here provide a significant improvement over the state of the art, and make in vivo, automated assessment of corneal endothelium more accessible. The segmentation method proposed paves the way to many possible new morphometric parameters, which can quickly and precisely be determined from the segmented image.

Cataract surgery in patients with Fuchs' endothelial corneal dystrophy: when to consider a triple procedure.

PURPOSE: To ascertain preoperative and intraoperative factors that predict the need for endothelial keratoplasty (EK) in patients with Fuchs' endothelial corneal dystrophy (FECD) undergoing cataract surgery. DESIGN: Prospective, observational cohort study. PARTICIPANTS: Eighty-nine patients (89 eyes) with FECD who require cataract surgery. METHODS: One month before cataract surgery, we assessed best-corrected visual acuity, contrast sensitivity, straylight, keratometry, ultrasonic pachymetry, intraocular pressure, 7 corneal features of FECD and cataract density at slit-lamp examination, and corneal backscatter using in vivo confocal microscopy (IVCM; Confoscan 4, NIDEK Technologies, Padova, Italy). After surgery, measurements were repeated at 1, 2, and 12 months. We used stepwise binary logistic regression analysis to evaluate 30 preoperative and 5 intraoperative parameters for their ability to predict the postoperative need for EK. Receiver operating characteristic (ROC) curves of the predictive factors were used to identify their optimal cutoff points. MAIN OUTCOME MEASURES: Central corneal thickness (CCT) and backscatter at the basal epithelial cell layer (EV). RESULTS: After cataract surgery, 35 (39%) of 89 eyes underwent EK to restore vision. Of all preoperative and intraoperative parameters, only CCT and EV were identified as significant factors, predictive of the need for EK. The area under the ROC curve of EV was significantly higher than that of CCT (P = 0.003), whereas a combination of both factors in a linear discriminant function did not improve the predictive value (P = 0.66). As optimal cutoff points, we chose 1894 scatter units for EV and 630 µm for CCT. Both cutoff points correspond with a specificity of 94% and represent sensitivity of 63% for EV and 40% for CCT. CONCLUSIONS: Backscatter at the basal epithelial cell layer measured by IVCM predicts the need for EK after cataract surgery in patients with FECD. As an indicator for the corneal hydration state, the EV improves patient selection for combined cataract surgery and EK. In deciding whether to perform a triple procedure, CCT remains a less effective, but adequate, alternative. Regardless of the predictive factor used, a tailor-made approach is recommended accounting for individuals' expectations.

Self-limiting corneal edema with multiple parallel lines on the endothelium (SCEMPLE).

The purpose of this study was to report the natural course and in vivo confocal microscopy (IVCM) findings of five cases with unilateral self-limiting corneal edema and multiple parallel lines on the endothelium (SCEMPLE). This study is an observational case series. Five patients, who experienced a blurred vision due to SCEMPLE, were studied using slit-lamp examination and white-light IVCM (Confoscan 4; Nidek Technologies, Padova, Italy). IVCM of the linear deposits revealed characteristic hyperreflective material protruding between the endothelial cells. The lines also displayed spot-like holes and polygonal precipitates, which resembled dislodged endothelial cells. Because other signs of corneal inflammation, such as stromal infiltration and ciliary injection, were lacking, the condition was left untreated. After 1 day, slit-lamp examination and IVCM revealed only a small residual of the lines. Spontaneous, complete resolution occurred in all five cases within 1 week, leaving a lower endothelial cell density of the affected eyes as only sequela. SCEMPLE requires no treatment but may result in endothelial cell loss. The self-limiting character and IVCM appearance dispute SCEMPLE being a form of endotheliitis and suggest a different etiology.

How normal is the transparent cornea? Effects of aging on corneal morphology.

PURPOSE: To ascertain the effects of aging on corneal morphology and to illustrate the morphologic diversity of the different layers in the normal cornea as seen by in vivo confocal microscopy (IVCM). DESIGN: Observational cross-sectional study. PARTICIPANTS: A total of 150 healthy subjects, evenly distributed over 5 age categories, comprising 75 men and 75 women. METHODS: Both transparent corneas (n = 300) of all subjects were examined in duplicate by white light IVCM (Confoscan 4, NIDEK Technologies, Albignasego, Padova, Italy). After reviewing the IVCM examinations for morphologic variations of the corneal layers, we selected the 8 most common features to illustrate the morphologic diversity. Subsequently, all 600 IVCM examinations were assessed for the presence of these features. We used binary logistic regression analyses to assess the age-relatedness of each feature. MAIN OUTCOME MEASURES: Age distribution of bright superficial epithelial cells, dendriform cells, alterations characteristic of epithelial basement membrane dystrophy (EBMD), tortuous stromal nerves, stromal microdots in the anterior stroma, folds in the posterior stroma, opacification of Descemet's membrane, and corneal guttae. RESULTS: Four features were found characteristic of the aging cornea: stromal microdots in the anterior stroma (P<0.0001), folds in the posterior stroma (P<0.0001), opacification of Descemet's membrane (P<0.0001), and corneal guttae (P<0.0001). Alterations characteristic of EBMD were found in 3% of all eyes and only detected in subjects aged ≥40 years, suggesting age-relatedness (P = 0.09). Other features, such as bright superficial epithelial cells (n = 38, 13%), dendriform cells (n = 42, 14%), and tortuous stromal nerves (n = 115, 38%), were age-independent. We also found a novel phenotype of corneal endothelium in 4 normal eyes of 2 subjects, which we coined "salt and pepper endothelium." We could not establish whether this novel phenotype represented a morphologic variant of normal endothelium, an early stage of a known corneal endothelial disorder, or a completely new disease entity. CONCLUSIONS: Knowledge of the common morphologic variations of the corneal layers and the effects of aging on corneal morphology as seen by IVCM increases our understanding of corneal degenerative disorders and is essential to detect corneal pathology. Our finding of a novel phenotype of corneal endothelium emphasizes the morphologic diversity of this optically transparent tissue.

Monitoring the inflammatory process in herpetic stromal keratitis: the role of in vivo confocal microscopy.

PURPOSE: To investigate the role of in vivo confocal microscopy (IVCM) in the detection of inflammatory activity and follow-up of herpetic stromal keratitis (HSK). DESIGN: Prospective observational cohort study. PARTICIPANTS: Thirty-eight patients with active HSK. METHODS: Within 7 days after diagnosis of active HSK, both eyes of each patient were examined by slit-lamp biomicroscopy and white-light IVCM (Confoscan 4; Nidek Technologies, Padova, Italy). The HSK-affected eyes were followed up at 1, 3, 6, and 12 months, whereas the unaffected fellow eyes were reexamined after 12 months. Three patients did not complete follow-up and were excluded for data analyses. All IVCM examinations were assessed for morphologic alterations characteristic of inflammatory activity and for corneal backscatter. As secondary outcome parameters, best-corrected visual activity (BCVA), central corneal thickness (CCT), intraocular pressure (IOP), and endothelial cell density (ECD) were determined at each study visit. We used repeated-measures analysis of variance to assess changes during the 12-month follow-up period and paired t tests to compare HSK-affected eyes with fellow eyes. MAIN OUTCOME MEASURES: Presence of dendriform cells, pseudoguttae, and keratic precipitates, and follow-up of mean corneal backscatter. RESULTS: An increase of dendriform cells and pseudoguttae often accompanied stromal infiltration. Because these IVCM parameters were indiscernible or overlooked at slit-lamp examination, they proved to be excellent indicators of inflammatory activity. At 12 months' follow-up, mean corneal backscatter had decreased significantly by 36%, but still fell outside the normal range in 24 (69%) of the HSK-affected eyes. By using slit-lamp in conjunction with IVCM, we detected 17 recurrences in 14 of 35 patients (40%). Three of these recurrences were missed by slit-lamp, and 6 of these were missed by IVCM. At 12 months' follow-up, BCVA (-9 letters), CCT (-36 µm), and ECD (-313 cells/mm(2)) were significantly lower, whereas IOP (1.8 mmHg) was significantly higher, in HSK-affected eyes compared with fellow eyes. CONCLUSIONS: The data presented demonstrate that IVCM is complementary to slit-lamp examination in the follow-up of HSK, particularly because of its power to detect early signs of intracorneal inflammatory activity. Therapy guidance based on morphologic assessment and corneal backscatter measurement by combined IVCM and slit-lamp examination may improve the outcome of HSK. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Zipper cell endotheliopathy: a new subset of idiopathic corneal edema.

PURPOSE: To report the clinical and histologic findings of a new subset of idiopathic corneal edema: zipper cell endotheliopathy. DESIGN: Observational case report. PARTICIPANT: A 55-year-old woman with unilateral bullous keratopathy. METHODS: Clinical observation consisted of slit-lamp examination and in vivo confocal microscopy (IVCM). Aqueous humor samples and the excised corneal button were analyzed for the presence of herpes viruses. The excised cornea was subjected to detailed immunohistochemistry (IHC) and scanning and transmission electron microscopy. MAIN OUTCOME MEASURES: Clinical and pathologic characteristics of zipper cell endotheliopathy. RESULTS: In vivo confocal microscopy revealed unique morphologic alterations of the corneal endothelial layer. Focal areas of denudation were surrounded by endothelial cells with zipper-like cell borders and intercellular structures. Besides central corneal edema, no other signs of corneal inflammation were detected. A herpes virus origin for the bullous keratopathy was excluded. The IHC analysis disclosed positive staining for cytokeratin (CK) 7, CK8/18, and CK19, suggesting epithelial metaplasia of the endothelial cells. Ultrastructural examination confirmed the IVCM findings by showing large areas of endothelial denudation and vacuolated endothelial cells with large, broad-based extensions that partially overlapped neighboring cells. Despite extensive complementary research and review of the literature, the endothelial alterations could not be attributed to any known corneal disorder. CONCLUSIONS: To the authors' knowledge, zipper cell endotheliopathy is a new subset of idiopathic corneal edema. The case report presented illustrates the potential use of IVCM to differentiate the spectrum of corneal disorders and to discover new corneal diseases.

Endothelial involvement in herpes simplex virus keratitis: an in vivo confocal microscopy study.

PURPOSE: To describe the appearance, frequency, and clinical consequences of corneal endothelial involvement in human herpes simplex virus (HSV) keratitis as seen by in vivo confocal microscopy (IVCM). DESIGN: Prospective observational case series. PARTICIPANTS: A total of 285 patients with HSV keratitis who visited the cornea department of the Rotterdam Eye Hospital between May 2005 and May 2008. The control groups comprised the unaffected fellow eyes of patients with HSV keratitis, the eyes of 58 healthy volunteers, and the affected eyes of 62 patients with inflammatory corneal disorders other than HSV. METHODS: We examined the eyes of all participants by IVCM and slit-lamp examination. For IVCM, corneas were scanned with Confoscan 3 or 4 (Nidek Technologies, Albignasego, Padova, Italy). MAIN OUTCOME MEASURES: All IVCM examinations were qualitatively reviewed for signs of endothelial deviations characteristic of endotheliitis. Endothelial cell density (ECD) was evaluated on the first and last visits of patients who were followed for more than 100 days. The differences in ECDs were calculated and converted to percent ECD change per year. RESULTS: Endothelial alterations characteristic of endotheliitis were detected by IVCM in 107 of 250 patients with HSV keratitis (43%). These deviations consisted of pseudoguttata, enlarged intercellular gaps, infiltration of inflammatory cells into the endothelial layer, loss of defined cell boundaries, spot-like holes, and endothelial denudation. All of these signs disappeared with appropriate antiviral and anti-inflammatory treatment. However, the endothelium in eyes with endotheliitis-characteristic alterations showed a significant decrease in ECD (10.3% per year) compared with healthy fellow eyes. CONCLUSIONS: IVCM allows earlier detection of endothelial alterations in patients with HSV keratitis compared with slit-lamp examination. Although endotheliitis-specific alterations appear to resolve, the corneal endothelium can become irreversibly damaged.

Graft insertion during Descemet-stripping automated endothelial keratoplasty: pulling the graft inward.

We describe a technique to facilitate insertion of the folded donor graft during Descemet-stripping automated endothelial keratoplasty (DSAEK). Surgery is performed using a standard technique, and the graft is pulled into the anterior chamber using a double-armed 10-0 polypropylene suture with straight 16 mm needles. The technique of pulling the graft inside the anterior chamber is easy and ensures that the graft is inserted and unfolded in the right direction. There is no compression of tissue so mechanical trauma to the endothelium is less than when an inserting forceps is used. The results in 12 cases are presented.

"Salt and Pepper Endothelium" Recurring After Descemet Membrane Endothelial Keratoplasty.

PURPOSE: To describe the presence of "salt and pepper endothelium", that is, typical cellular inclusion bodies in a patient with Fuchs endothelial corneal dystrophy (FECD), that recurred in the donor corneal endothelial cells after Descemet membrane endothelial keratoplasty (DMEK). METHODS: A 76-year-old man underwent DMEK for FECD in his left eye. Routine specular microscopy imaging, best-corrected visual acuity measurements, and pachymetry measurements were performed before and after surgery. RESULTS: Besides large guttae indicating FECD, preoperative specular microscopy images showed variable-sized dark cellular inclusion bodies in the endothelial cells. One month after DMEK, donor endothelial cells appeared normal; however, at 3 months, the typical inclusion bodies reappeared and progressed slowly within a 4-year follow-up period. Both best-corrected visual acuity and pachymetry were stable throughout the study period. CONCLUSIONS: "Salt and pepper endothelium" recurred after the host tissue was exchanged by donor Descemet membrane, that is, a DMEK graft. These changes may indicate that either the donor corneal endothelial cell morphology is modified by adjacent tissue structures or that it is completely replaced by recipient endothelium within the first months after surgery.

Descemet stripping automated endothelial keratoplasty: effect of intraoperative lenticule thickness on visual outcome and endothelial cell density.

PURPOSE: To determine the correlation between the intraoperative donor lenticule thickness in Descemet stripping automated endothelial keratoplasty (DSAEK) and both the best-corrected visual acuity (BCVA) and endothelial cell density (ECD) at 6 months. To describe relevant surgical considerations with regard to the choice of microkeratome head. DESIGN: Prospective case series. METHODS: Thirty-five patients (37 eyes) undergoing DSAEK were included in this prospective case series. Intraoperative donor lenticule thickness, assessed by ultrasound pachymetry, was compared with BCVA and ECD 6 months postoperatively. RESULTS: Mean BCVA logarithm of the minimal angle of resolution (logMAR) was 0.58 preoperatively and 0.29 6 months postoperatively. Three eyes were excluded from BCVA analysis. The average donor lamellae thickness was 175 µm. Mean ECD loss was 39% 6 months postoperatively compared with that preoperatively. There was no correlation between donor lamella pachymetry and BCVA logMAR and ECD at 6 months in this series (P = 0.13 and P = 0.09, respectively). Analogous to our findings, a review of published reports on donor pachymetry in DSAEK did not provide data to suggest a correlation between donor corneal thickness and postoperative BCVA or ECD. CONCLUSIONS: Donor lenticule thickness does not have a direct effect on 6-month postoperative visual acuity or endothelial cells. Efforts should be made to reduce surgical trauma to the lenticule rather than adhering to a strict depth when cutting the graft. We propose using the Moria 350 µm microkeratome head for corneal thickness more than 600 µm and the 300 footplate for corneal thickness less than 600 µm.

Normative database for corneal backscatter analysis by in vivo confocal microscopy.

PURPOSE: To ascertain the sex and age relatedness, diurnal variation, and repeatability of backscatter measurement in the normal human cornea. METHODS: Seven corneal backscatter variants were measured by in vivo confocal microscopy (IVCM) in both normal eyes (n = 314) of 157 healthy subjects. These subjects were assigned to one or more of three groups. The sex and age relatedness of corneal backscatter were assessed in group 1 (n = 300), which comprised 75 men and 75 women evenly distributed over five age categories. To assess diurnal variation, eyes in group 2 (n = 40) were measured four times a day, at 3-hour intervals. The eyes in group 3 (n = 50) were examined four times a year to determine intersession repeatability. Intrasession repeatability was determined by performing all IVCM examinations in duplicate. Linear mixed models were used to assess the effects of sex, age, and time of measurement on corneal backscatter. RESULTS: Mean corneal backscatter was 3.5% higher in men (P = 0.003). From the age of 50 years, backscatter increased significantly in the anterior stroma (P = 0.0003). A small but statistically significant diurnal variation was found in all seven backscatter variants (P < 0.01). The test-retest coefficient of variation of mean corneal backscatter was 5.3%, comprising intra- and intersession repeatability. CONCLUSIONS: Sex and time of measurement significantly affect corneal backscatter measured by IVCM, whereas age affects only backscatter in the anterior stroma. All three factors should be taken into account when conducting scientific research. For ophthalmic practice, the authors suggest ignoring these factors and propose a generalized normal range and minimum detectable change for each backscatter variant.

Wide-range calibration of corneal backscatter analysis by in vivo confocal microscopy.

PURPOSE: To report intra- and interinstrument calibration methods for corneal backscatter analysis by in vivo confocal microscopy. METHODS: Applicability of two reference standards was evaluated for corneal backscatter calibration. Repeated measurements of four concentrations of AMCO Clear (GFS Chemicals, Inc., Powell, OH) suspension and three transparencies (26%, 49%, and 65%) of polymethylmethacrylate (PMMA) slabs were performed to assess image intensity acquisition in a wide backscatter range. Intra- and intersession repeatability and lot-to-lot variation were determined for both standards. The effect of light intensity (LI) variation on image intensity acquisition was evaluated by examination of PMMA slabs with nonreference (60% and 80%) and reference (72%) LIs. Both reference standards were implemented in the protocol. Intrainstrument calibration was verified by measuring three normal corneas with 60%, 72%, and 80% LIs. Interinstrument calibration was tested by measuring PMMA slabs on a second, similar confocal microscope. RESULTS: AMCO Clear was used to express image intensity in absolute scatter units (SU), whereas the 49% transparent PMMA slab showed best repeatability, without image saturation, to adjust for LI variation. Intrainstrument calibration for LI variation reduced mean differences from -38.3% to 1.7% (60% LI) and from 33.9% to -0.6% (80% LI). The mean difference between similar microscopes decreased from 18.4% to 1.2%, after calibration of the second microscope. CONCLUSIONS: Large interinstrument differences necessitate calibration of corneal backscatter measurements. With AMCO Clear suspension and PMMA slabs, standardization was achieved in a wide backscatter range corresponding to normal and opaque corneas. These methods can easily be applied in ophthalmic practice.

Long-term follow-up of hydrogel intracorneal lenses in 2 aphakic eyes.

We report the outcome of hydrogel intracorneal lens implantation in 2 patients. The lenses were implanted at approximately 50% depth in the cornea to correct high hyperopic refractive errors of 10.5 diopters (D) and 14.0 D, respectively. Both patients were contact lens intolerant and not suitable for intraocular lens implantation. Surgery was performed in 1988, and the patients were followed until early 2010. The patients showed good tolerance for the intracorneal lenses, but both developed opacities around the implant, leading to reduced visual acuity in 1 patient. Long-term patient monitoring is essential since corneal opacities can develop after many years. Removing the implant is not necessary as the lens can easily be rinsed by lifting the corneal cap.