Corneal Impression Cytology Research Articles

Cytokeratin 13 Is a New Biomarker for the Diagnosis of Limbal Stem Cell Deficiency.

The purpose of this study was to evaluate the expression of cytokeratin (K) 13 on the corneal surface and to validate its application in the diagnosis of limbal stem cell deficiency (LSCD). This prospective comparative study included 26 corneal impression cytology (IC) specimens from patients diagnosed with LSCD. Twenty-three IC specimens from normal donors served as controls. K12 and K13 expression were detected on the IC specimens by immunohistochemistry study. The number of K12 + or K13 + cells in all areas of the IC was quantified using ImageJ software. The epithelial cells harvested from IC specimens from control corneas were all K12 + . In eyes with LSCD, K13 + and K12 + /K13 + cells accounted for 93.8% and 2.6%, respectively, in the cornea. In eyes with sectoral LSCD, the median number of K13 + cells in the clinically affected area was higher than that in the unaffected area (810.0 vs. 115.0 cells/mm 2 ; P < 0.001). No significant correlation was found between the LSCD severity and the number of K12 + cells (r = -0.284, P = 0.16) or K13 + cells (r = -0.011, P = 0.95). The presence of at least 16 K13 + cells/mm 2 was suggestive of LSCD. Identification of K13 + cells on IC specimens provides a simple and reliable method to detect conjunctival epithelial cells on the cornea. K13 is a marker for diagnosing LSCD and localizing the involved area in sectoral LSCD.

Cytokeratin 13 by Immunofluorescence Analysis is an Adjunct Marker for the Diagnosis of Limbal Stem Cell Deficiency on Corneal Impression Cytology Samples

Cytokeratin 13 by Immunofluorescence Analysis is an Adjunct Marker for the Diagnosis of Limbal Stem Cell Deficiency on Corneal Impression Cytology Samples

Evaluation of ocular surface status in patients with glaucoma treated with preservative or preservative-free prostaglandin analogs

Background Glaucoma is one of the eye diseases that require chronic treatment. Prostaglandin derivatives are popular for treating glaucoma. Most prostaglandin drops contain benzalkonium, which contains the contraceptive pill. Aim Comparing the ocular surface changes in patients with primary open-angle glaucoma treated with preservative-free versus preserved prostaglandin analogs (PGAs). Patients and methods This prospective study was carried out on 40 eyes of 40 patients with newly diagnosed primary open-angle glaucoma and randomly divided into two groups that received PGAs: group A (20 eyes) received preserved PGA (latanoprost 0.005%), and group B (20 eyes) received preservative-free PGA (tafluprost 0.0015%). After 3 months of treatment, the ocular surface status in every patient was evaluated by Schirmer 1 test, tear film break-up time, fluorescein corneal staining, impression cytology, and Ocular Surface Disease Index questionnaire. Results Schirmer 1 test showed that 90% of patients in group B had normal values compared with 40% in group A (P=0.008). Tear film break-up time showed that 90% of patients in group B had normal values compared with 35% in group A (P=0.004). Regarding fluorescein corneal staining, 100% of the patients in group B had normal values compared with 60% in group A (P=0.007). Impression cytology grading was significantly better in group B (P&lt;0.001). According to the Ocular Surface Disease Index score, patients on tafluprost treatment reported better drug tolerability (P=0.041). Conclusion Patients with glaucoma treated with preservative-free PGA have fewer ocular surface adverse effects, which may increase the tolerability of topical glaucoma medications.

Changes in Corneal Expression of MUC5AC after Autologous Serum Eyedrop Treatment in Patients with Limbal Stem Cell Deficiency

ABSTRACT Purpose To evaluate the effect of autologous serum eyedrops treatment on corneal expression of the MUC5AC in patients with limbal deficiency. Methods A prospective and comparative interventional case series study of 42 eyes of 21 patients was performed before and 8 weeks after treatment with autologous serum. All patients underwent a complete ophthalmic assessment of the tear film and ocular surface, corneal impression cytology (IC) and MUC5AC detection by Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Results Forty-one of the 42 eyes studied were available for both conventional cytology and MUC5AC analysis prior to and after treatment. Differences between outcomes obtained by impression cytology and MUC5AC detection were found in 9 of 82 samples (11%). We found changes in the corneal expression of MUC5AC after treatment in 19 of 41 eyes (46.3%): 18 of them (94.7%) changed from positive to negative expression, and 1 eye (5.3%) changed from negative to positive MUC5AC expression after autologous serum eyedrops. These changes were related with the corneal involvement prior to treatment (15 of them (78.9%) occurred in patients with slight corneal involvement), and with the improvement in the degree of squamous metaplasia after treatment (P = .001 and P = .003, respectively). Conclusions The treatment significantly improved tear stability, squamous metaplasia, and subjective patient perception. Autologous serum eyedrops treatment diminished the corneal expression of MUC5AC mainly in patients with slight corneal involvement before treatment.

Outcomes of Penetrating Keratoplasty Following Autologous Cultivated Limbal Epithelial Stem Cell Transplantation.

The purpose of this study is to investigate the outcomes of penetrating keratoplasty (PKP) following autologous cultivated limbal epithelial stem cell transplantation (CLET). A prospective, single center, interventional cohort study investigating patients with unilateral total limbal stem cell deficiency (LSCD) treated with CLET who underwent PKP. Patients with confirmed corneal re-epithelialization > 6 months post-CLET, and with best-corrected visual acuity (BCVA) <0.3 logMAR were offered PKP. CLET survival assessed by slit lamp, corneal impression cytology (CIC), and in vivo confocal microscopy. Confirmation of corneal re-epithelialization by histological and immunocytochemical (ICC) examination of trephined corneal buttons. Mean change in best-corrected visual acuity (logMAR) following PKP and PKP survival at 12 months were calculated. Twenty patients underwent PKP. Mean time of PKP was 19 months (range 11-41 months, SD 7.26) post-CLET. Median follow-up time post-PKP was 15 months (range 1-32, SD 10.2). CIC and ICC of all corneas confirmed corneal re-epithelialization before PKP. Mean pre-PKP BCVA was 1.46 (range 0.3-2.7, SD 0.94) improving to a mean post-PKP BCVA of 0.74 (range 0-2.7, SD 0.87); mean improvement in BCVA post-PKP of 36 letters (95% CI 15.0-57.1, p = .002). Kaplan-Meier mean graft survival was 90.9% (95% CI 50.8-98.7) at 12 months. We recommend a two-stage approach with CLET followed by PKP >12 months later. Patients experienced a significant improvement in BCVA following PKP. PKP did not have a detrimental effect on CLET survival. PKP survival post-CLET is better than that reported for high risk PKP. Stem Cells 2018;36:925-931.

Clinical features and corneal optical anisotropies in a rabbit model of limbal stem cell deficiency.

To investigate the intra-laboratory reproducibility of clinical features and to evaluate corneal optical anisotropies in a rabbit model of limbal stem cell deficiency. Limbal injury was induced in the right eye of 23 adult New Zealand White rabbits using a highly aggressive protocol that combined 360 degrees limbal peritomy, keratolimbectomy, alkaline chemical burn, and mechanical removal of the epithelium. Clinical evaluation of the injured eyes was performed for 28 days and included corneal impression cytology. Corneas with a severe clinical outcome set typical of limbal stem cell deficiency were then collected, subjected to a histopathological examination, and examined for optical anisotropies. Corneas from healthy rabbit eyes were used as controls. Differences in optical path due to stromal collagen birefringence, as well as linear dichroism related to the expression and spatial orientation of glycosaminoglycan chains from proteoglycans, were measured from cross-sections under a quantitative polarized light microscope. One eye showed signs of hypopyon and was excluded. Signs of ocular inflammation were observed in all eyes studied (n=22). Corneal impression cytology did not detect goblet cells. Twelve of the 22 corneas presented a clinical outcome set typical of limbal stem cell deficiency, which is characterized by the presence of epithelial defects, inflammatory cells, moderate-to-severe opacity, and neovascularization. Microscopic studies under polarized light revealed that relative to controls, limbal stem cell deficiency caused a 24.4% increase in corneal optical path differences. Further, corneas with limbal stem cell deficiency were less dichroic than controls. These results suggest that rabbit models of limbal stem cell deficiency must be rigorously screened for use in preclinical studies to ensure experimental homogeneity because protocols used to create limbal stem cell deficiency could be not associated with good intra-laboratory reproducibility of clinical features. Limbal stem cell deficiency, as induced herein, altered the optical anisotropic properties of the corneal stroma. Such alterations are indicative of changes in collagen packing and the spatial orientation of glycosaminoglycan chains from proteoglycans. Knowledge of these changes is important to potentiate strategies aimed at restoring the morphofunctional integrity of the corneal stroma affected by limbal stem cell deficiency.

Monocarboxylate Transporters Mediate Fluorescein Uptake in Corneal Epithelial Cells.

To determine the presence of monocarboxylate transporter (MCT) in human and rabbit corneal epithelium and its role in transcellular fluorescein transportation in the cornea. The presence of MCTs in human and rabbit corneal epithelium was determined by RT-PCR and immunohistochemistry. Intracellular fluorescein uptake experiment was performed using cultured human corneal epithelial cells (HCECs). The involvement of MCT in fluorescein uptake was determined by addition of MCT inhibitors to HCECs and acute dry eye model on New Zealand albino rabbits by spectrophotometry, corneal impression cytology, and external eye photographs. MCT-1 and MCT-4 were identified in both human and rabbit corneal epithelia. A longer treatment period and a lower pH value in culture medium increased fluorescein uptake in HCECs. Fluorescein uptake in HCECs was decreased following addition of MCT inhibitors in a concentration-dependent manner. Impression cytology under fluorescent microscopy showed intracellular fluorescein staining in the rabbit cornea with acute dry eye treatment that was decreased following topical treatment of MCT inhibitors. Fluorescein ingress in corneal epithelial cells is mediated by the MCT family. Further study of MCT-mediated transport on HCECs may potentially benefit differential diagnosis and contribute better understandings of ocular surface disorders.

Corneal Impression Cytology for the Diagnosis of Limbal Stem Cell Deficiency in a Dog

Corneal Impression Cytology for the Diagnosis of Limbal Stem Cell Deficiency in a Dog

Comparison of treatment with preservative-free versus preserved sodium hyaluronate 0.1% and fluorometholone 0.1% eyedrops after cataract surgery in patients with preexisting dry-eye syndrome

To compare treatment with preservative-free and preserved sodium hyaluronate 0.1% and fluorometholone 0.1% eyedrops after cataract surgery in patients with preexisting dry-eye syndrome. Bucheon St. Mary's Hospital, Catholic University of Korea, Seoul, Korea. Randomized controlled study. Patients with cataract and dry-eye syndrome were randomly divided into 2 groups. Group 1 patients were treated with preservative-free sodium hyaluronate 0.1% and preservative-free fluorometholone 0.1% eyedrops 4 times a day in the first month and twice a day in the second month. Group 2 patients were treated with preserved eyedrops using the same schedule. Ocular Surface Disease Index (OSDI) score, tear-film breakup time (TBUT), Schirmer I test, corneal fluorescein staining, impression cytology, and antioxidant and inflammatory cytokine activities in tears were evaluated. Both groups comprised 40 patients. At 2 months, the OSDI score, TBUT, Schirmer I score, fluorescein staining score, impression cytology findings, and goblet cell count were significantly better in Group 1 than in Group 2 (P<.05). The interleukin-1β and tumor necrosis factor-α concentrations were significantly less in the tears of Group 1 patients than in the tears of Group 2 patients, and catalase and superoxide dismutase 2 fluorescence intensities were significantly greater in the tears of Group 1 patients than in the tears of Group 2 patients (P<.05). Preservative-free sodium hyaluronate 0.1% and fluorometholone 0.1% eyedrops can improve the symptoms and signs of dry-eye syndrome after cataract surgery. Preservative-free fluorometholone eyedrops may have antiinflammatory and antioxidant effects in tears of patients with dry-eye syndrome. No author has a financial or proprietary interest in any material or method mentioned.

Age-related alterations in the lacrimal gland of adult albino rat: A light and electron microscopic study

BackgroundAge related changes in the lacrimal gland are associated with alterations in the structural organization and functional response in the gland of diverse mammalian species. Dry eye syndrome is one of the most common ocular problems in the world especially in old age. It results when the lacrimal gland fails to secrete proteins and fluid in sufficient quantity or appropriate composition. Aim of the workThe present study is designed to demonstrate the influence of aging on the structure of the lacrimal gland of albino rat and to provide a morphological basis to explain the pathogenesis of the dry eye syndrome with ageing. It also aims to carry out a comparative analysis of age-dependent changes in male and female rats and to address how the lacrimal gland ages in each sex. Material and MethodsEighty albino rats were used in this study. The animals were divided into two age groups, young adult and senile. Tear secretion was measured using a modified Schirmer test. Corneal impression cytology of the anesthetized rats was done. The glands were subjected to gross morphologic examination, microscopic examination using H&E, PAS, Masson's trichrome and Giemsa stains. Electron microscopic examination was done in addition to quantitative histomorphometric estimations included acinar density, ductal count and mast cell count. ResultsLight microscopic examination of the lacimal glands of the senile rats revealed different pathological changes. These included acinar, ductal as well as stromal changes. Electron microscope examination of the lacrimal gland of the senile group showed a decrease in the electron dense secretory vesicles, mitochondrial swelling and lipofuscin-like inclusions were frequently seen in the cytoplasm of acinar cells in senile rats. ConclusionThe structural changes in the lacrimal glands of senile rats were associated with reduction in tear secretion as well as alterations in corneal epithelium. Gender difference in lacrimal gland structure was recorded.

Impression cytology and in vivo confocal microscopy in corneas with total limbal stem cell deficiency

To describe corneal changes seen on in vivo confocal microscopy in patients with total limbal stem cell deficiency and to correlate them with cytological findings. A prospective case series including 13 eyes (8 patients) with total limbal deficiency was carried out. Stem cell deficiency was diagnosed clinically and by corneal impression cytology. Confocal images of the central cornea were taken with the Heidelberg Retina Tomograph II, Rostock Corneal Module (Heidelberg Engineering, Heidelberg, Germany). Impression cytology of the cornea revealed conjunctival epithelial cells and goblet cells in all cases. In vivo confocal microscopy showed disruption of normal layers of the corneal epithelium in all eyes. Confocal images showed cells with characteristics of conjunctival epithelium at the cornea in 76.9% of the total. These findings on confocal microscopy were compatible to limbal stem cell deficiency. Additionally, goblet cells, squamous metaplasia, inflammatory cells and dendritic cells were observed. The sub-basal nerve plexus was not identified in any of the corneas. Corneal neovessels were observed at the epithelium and stroma. All cases showed diffuse hyper-reflective images of the stroma corresponding to opacity of the tissue. Limbal stem cell deficiency had been confirmed by impression cytology in all cases, and 76.9% of the cases could also be diagnosed by in vivo confocal microscopy through the conjunctival epithelial cell visualization on the corneal surface. Frequent confocal microscopy findings were abnormal cells at the cornea (conjunctival epithelial, goblet and inflammatory cells), corneal neovessels and diffuse hyper-reflection of the stroma.

A Mouse Model of Limbal Stem Cell Deficiency Induced by Topical Medication With the Preservative Benzalkonium Chloride

To develop a mouse model of limbal stem cell deficiency (LSCD) by topical administration of benzalkonium chloride (BAC). BAC solutions (0%-0.5%) were applied to the mouse ocular surface for 4 weeks. Corneal neovascularization, inflammation, and epithelial status were observed under slit-lamp microscope. The eyeball and ocular surface tissues were collected at 4 and 12 weeks and labeled with a series of antibodies. Limbal structure was evaluated by light and transmission electron microscopy (TEM). Corneal impression cytology was performed at 12 weeks, and specimens were labeled with periodic acid Schiff (PAS) reagents. BAC (0.5%) four times per day for 28 days successfully induced the typical manifestations of LSCD, including corneal neovascularization, severe inflammation in the stroma, and diffuse epithelial defect (P < 0.001). Conjunctival epithelium markers K19 and K13 were positive on the corneal surface. Expression of the putative limbal stem cell markers P63 and ABCG2 was abolished in the limbal epithelium. β-catenin was negative in the basal layer. TEM revealed the irregular basement membrane and the loss of stem cell-specific ultrastructure in the limbal basal epithelium. In the 0.5% BAC group, goblet cells could not be observed on day 28 but emerged after the cessation of BAC, and remained over the cornea after 8 weeks. K13-positive cells were still present over the cornea with the loss of K12. Topical administration of BAC at high concentration and frequency in mouse induces ocular surface changes resembling those of LSCD in humans, representing a novel model of LSCD.

Novel Molecular Diagnostic System of Limbal Stem Cell Deficiency Based onMUC5ACTranscript Detection in Corneal Epithelium by PCR-Reverse Dot Blot

To evaluate the sensitivity and specificity of a PCR-strip system based on reverse dot blot for detection of MUC5AC mRNA in corneal epithelium samples from patients with limbal stem cell deficiency (LSCD), and to determine the correlation with clinical diagnosis. We obtained 87 corneal impression cytology (IC) samples from 55 subjects (37 patients clinically diagnosed with LSCD and 18 control subjects). Total RNA was extracted from each IC sample and retrotranscribed to cDNA. MUC5AC mRNA transcript was amplified by a customized RT-PCR assay and detected in PCR strips based on reverse dot blot hybridization and in agarose gels. Conjunctival IC samples were used as positive controls. Forty-four of 45 corneal IC samples obtained from patients clinically diagnosed with LSCD were positive for MUC5AC, whereas 34 of 42 corneal ICs from healthy subjects were negative for MUC5AC. Four healthy corneas were found MUC5AC positive, and four rendered inconclusive results. A correlation of 91.4% (P < 0.001) between molecular testing and clinical diagnosis was found. The overall sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the PCR-strip system were 98%, 89%, 92%, and 97%, respectively. Corneal epithelium MUC5AC transcript detection by reverse dot blot PCR-strip is a highly sensitive technique for LSCD diagnosis. The test results strongly correlate with clinical diagnosis of characterized LSCD cases. The PCR-strip system may be used for early detection, and for the detection of mild cases of LSCD, and constitutes an objective clinical tool for the monitoring of treatments and surgical decisions.

Clinical and Cytologic Evidence of Limbal Stem Cell Deficiency in Eyes With Long-Standing Vernal Keratoconjunctivitis.

We aimed to study the impression cytology (IC) of the ocular surface in eyes with vernal keratoconjunctivitis (VKC) and clinical evidence of limbal stem cell deficiency (LSCD). This is a prospective comparative study. This study included 78 eyes of 40 patients with VKC. Limbal stem cell deficiency was diagnosed clinically based on the presence of corneal findings such as dull irregular epithelial reflex, superficial neovascularization, conjunctivalization, and loss of limbal palisades of Vogt. The study group consisted of 28 eyes of 15 patients with clinically diagnosed LSCD and control group of 50 eyes of 25 patients without LSCD. Conjunctival and corneal IC was done in all eyes. Presence of goblet cells in the corneal samples on IC was considered confirmatory of LSCD. Compared with controls, patients with LSCD were older and had longer duration of disease. On IC, goblet cells were present on the cornea in 53.6% of eyes with clinically diagnosed LSCD and in none of the control eyes (P < 0.0001). Clinically diagnosed LSCD in study eyes correlated with cytologic findings of greater conjunctival squamous metaplasia, decreased conjunctival goblet cells, greater corneal cell metaplasia, and increased inflammation as compared with control eyes. Most of the eyes with VKC and clinical evidence of LSCD have cytologic evidence of LSCD with goblet cells on the cornea.

Evaluation of limbal stem cell deficiency: a new diagnostic tool based on keratin 13 immunostaining in corneal impression cytology

AbstractPurpose The aim of this study was to develop a validated, reliable and minimally invasive technique for diagnosing limbal stem cell deficiency (LSCD) using immunocytochemical detection of conjunctival and corneal keratins on epithelial cells collected by impression cytology (IC).Methods After validation of the labelling techniques on a cohort of ten healthy control patients, keratins K12, K13 and K19 were labelled on corneal IC of ten eyes suspected of LSCD. Positive scores for the conjunctival markers K13/K19, combined with the rarity of the corneal marker K12, were diagnostic proof of LSCD.Results IC is a reliable and noninvasive technique for collecting epithelial cells. The labelling validation phase eliminated K3 labelling due to lack of corneal specificity. Among the patients with suspected LSCD, nine samples were diagnosed with LSCD, which was severe (K12 negative) in eight cases and mild (K12 positive) in one case. One sample could not be analysed due to insufficient cells.Conclusion The immunocytochemical search for the K13/K19 pair using corneal IC provides a simple and reliable method for diagnosing LSCD, while the level of K12 provides a score of disease severity. On the other hand, we question the corneal specificity of K3 as conventionally established. Finally, to our knowledge, this is the first study detecting K13 on a conjunctival IC specimen as a marker of conjunctival differentiation.

Trehalose treatment accelerates the healing of UVB-irradiated corneas. Comparative immunohistochemical studies on corneal cryostat sections and corneal impression cytology.

The UVB-irradiated cornea is damaged by oxidative stress. Toxic oxygen products induced by UVB radiation in the cornea are insufficiently removed by antioxidants, whose numbers decrease with increasing UVB irradiation. In addition, the UVB-irradiated cornea suffers from hypoxic conditions because damaged corneal cells cannot utilize oxygen normally, although the supply of oxygen to the cornea is unchanged (normal). This contributes to attenuated re-epithelialization, corneal neovascularization and apoptotic cell death. Our previous publications reported that trehalose applied on the corneal surface during irradiation significantly suppressed UVB-induced corneal oxidative damage. The results of this study provide for the first time important evidence that trehalose applied on the surface of corneas for two weeks following repeated UVB irradiation (312 nm, daily dose 0.5 J/cm2) accelerated corneal healing, restored corneal transparency and suppressed corneal neovascularization. Compared to buffered saline treatment, following which caspase-3, nitrotyrosine, malondialdehyde and urokinase-type plasminogen activator were still strongly expressed in the corneal epithelium two weeks after irradiation and corneal neovascularization was evident, apoptotic cell death was already significantly reduced after one week of trehalose application. The expression of other markers of injury returned to normal levels during two weeks of trehalose treatment. In conclusion, our results show that trehalose accelerated healing of the UVB irradiated cornea, very probably via suppression of hypoxia-response injury. In addition, immunohistochemical results on corneal cryostat sections corresponded with those obtained using corneal impression cytologies, thus confirming that corneal impression cytologies are useful for diagnostic purposes.

Comparative Study of Limbal Stem Cell Deficiency Diagnosis Methods: Detection of MUC5AC mRNA and Goblet Cells in Corneal Epithelium

To evaluate a limbal stem cell deficiency (LSCD) diagnosis method based on the detection of the MUC5AC transcript by reverse transcription-polymerase chain reaction (RT-PCR) in comparison with the standard diagnostic method based on goblet cell detection by periodic acid-Schiff (PAS)-hematoxylin staining, using samples obtained from corneal epithelium impression cytology (IC). Transversal, comparative case series. We studied 59 eyes from 43 patients clinically diagnosed with LSCD. Impression cytology was used to gather cells from corneal and conjunctival epithelium from the same eye. The presence of goblet cells in the cornea was determined by PAS-hematoxylin staining, whereas the presence of the MUC5AC transcript was detected by RT-PCR using a custom-designed primer pair. Goblet cells in the corneal epithelium were detected by light microscopy, and the MUC5AC transcript was detected as the corresponding PCR amplicon in agarose gels. Our study included 59 corneal samples, together with their respective conjunctival samples for RT-PCR assays. Of these, 47 samples were also available for comparative PAS-hematoxylin staining. The MUC5AC amplicon was detected in 56 of 59 (94.9%) corneal epithelium samples. In contrast, conventional IC staining detected goblet cells in only 17 of 47 (36.2%) samples; these were not found in 27 of 47 (57.4%) samples (negative results), and 3 of 47 (6.4%) showed inconclusive results. The detection of the MUC5AC transcript in corneal epithelium is a more sensitive method to diagnose LSCD than the conventional PAS-hematoxylin method, although a minimum RNA concentration of 1.2 ng/μl is required for negative results to be reliable. Moreover, RT-PCR is a highly specific and more objective technique. Overall, these findings indicate that molecular analysis facilitates a more precise clinical diagnosis of LSCD, thereby reducing the risk of surgical failure.

Keratin 13 Immunostaining in Corneal Impression Cytology for the Diagnosis of Limbal Stem Cell Deficiency

The aim of this study was to develop a validated, reliable, and minimally invasive technique for diagnosing limbal stem cell deficiency (LSCD) by immunocytochemical detection of conjunctival and corneal keratins on epithelial cells collected by impression cytology (IC). After validation of labeling techniques on a cohort of 10 healthy control patients, keratins K12, K13, and K19 were labeled on corneal IC of 10 eyes suspected of LSCD. Positive scores for the conjunctival markers K13/K19, coupled with the rarity of the corneal marker K12, were diagnostic proof of LSCD. IC is a reliable and noninvasive technique for collecting epithelial cells. The labeling validation phase has permitted K3 labeling to be eliminated due to lack of corneal specificity. Among patients with LSCD, nine samples were diagnosed with LSCD (K13+/K19+), which was severe (K12-) in eight cases and mild (K12+) in one case. One sample could not be analyzed due to lack of cells. K13 has shown to be a new marker of conjunctival differentiation. The immunocytochemical search for the K13/K19 couple by corneal IC provides a simple and reliable method for diagnosing LSCD, whereas the level of K12 could provide a score of disease severity. On the other hand, the authors question the corneal specificity of K3 as conventionally established.

Increased Expression of Hepcidin and Toll-Like Receptors 8 and 10 in Viral Keratitis

Antimicrobial peptides (AMPs) and toll-like receptors (TLRs) form part of the "chemical barrier" of the ocular surface to microbes. Evidence suggests that pathogen recognition by TLR releases AMPs, altering AMP-TLR profiles in pathological states. This study investigated ocular surface expression of AMP-TLRs in health and disease. Complementary DNA from conjunctival and corneal impression cytology samples was used for semiquantitative and quantitative polymerase chain reactions, to determine gene expression of 6 AMPs and TLRs-1-10, in healthy subjects and patients with bacterial (n = 6), viral (n = 6), Acanthamoeba (n = 3), or dry eye (n = 7) diseases. Semiquantitative polymerase chain reaction showed variable AMP expression within groups and some expression patterns between groups, increased levels of LEAP (liver-expressed antimicrobial peptide)-1/hepcidin in viral disease, LEAP-2 in dry eye, and human beta defensin 3 in bacterial disease. There was no significant variability in TLR expression. Quantitative polymerase chain reaction showed significantly higher expression of LEAP-1 (P = 0.002) and TLR-8 (P = 0.023) and TLR-10 (P = 0.014) in viral keratitis and LEAP-2 (P = 0.034) in dry eye, versus controls. Increased expression of LEAP-1 and TLRs 8 and 10 in viral keratitis is novel; TLR-10 has not previously had a documented ligand. LEAP-2 may have a role in dry eye. Further studies will help to improve the understanding of these diseases and yield novel therapeutic interventions.

Diagnostics of dry eye

Dry eye is a multi-factorial disease of tears and ocular surface resulting in symptoms of discomforts, vision disabilities, unstable tear film and possible damage to the ocular surface. The final diagnosis of dry eye results from collecting various data obtained in the course of several diagnostic procedures. DRY EYE - DIAGNOSTIC METHODS: Diagnosis of dry eye involves performing a series of clinical methods and laboratory tests. This paper presents some of the procedures used in the diagnosis of dry eye. Biomicroscopy of the eye with objective tests to check the tear film (Schirmer I, rose bengal, time break up precorneal tear film), verification of corneal sensitivity and impression cytology of the conjunctiva, are the optimum dry eye diagnostic routine procedures. The combination of clinical and histopathological methods of examination of the anterior ocular surface is the basis for a quality and precise diagnosis of dry eye.