Piera Versura

Performance of Tear Osmolarity Compared to Previous Diagnostic Tests for Dry Eye Diseases

Purpose: Tear osmolarity is considered a key point in dry eye disease (DED) and its measurement is the gold standard in dry eye diagnosis. Tear osmolarity was evaluated in dry eye (DE) patients vs. a control group to assess its diagnostic performance compared to clinical and laboratory tests performed in either clinical or research settings. Methods: Tear osmolarity was measured with the TearLab Osmolarity System (OcuSense) in 25 normal subjects and 105 DE patients (severity score 1–4, Dry Eye Workshop (DEWS)). The following tests were also performed: Ocular Surface Disease Index (OSDI) symptoms questionnaire, Schirmer I test, Tear Film Break Up Time (TFBUT), ferning test, lissamine green staining, tear clearance, corneal esthesiometry, and conjunctival cytology by scraping and imprint. Statistical evaluation was performed by unpaired Student’s t and Mann-Whitney tests, the Spearman’s ρ and the Pearson’s r correlation coefficients (significance p < 0.05); all variables were also analyzed for sensitivity, specificity, Receiver Operating Characteristics (ROC) curves, likelihood ratio LR+, and positive predictive value (PPV). Results: Tear osmolarity normal values were 296.5 ± 9.8 mOsm/L, increasing values were shown stepwise DE severity (mild to moderate to severe dry eye, respectively: 298.1 ± 10.6 vs. 306.7 ± 9.5 vs. 314.4 ± 10.1, p < 0.05). A progressive worsening occurred in all the parameters with DED severity increase. Tear osmolarity exhibited the larger correlation strength vs. tear clearance, TFBUT and clinical score, strength increased with DED severity, mainly to inflammatory score and corneal sensitivity. Tear osmolarity 305 mOsm/L was selected as cut-off value for dry eye, 309 mOsm/L for moderate dry eye, 318 mOsm/L for severe dry eye (Area-Under-the-Curve was 0.737, 0.759, and 0.711, respectively). Conclusions: Tear osmolarity can now be considered a test suitable to be performed in a clinical setting. It showed a good performance in dry eye diagnosis, higher than the other tests considered, mainly in severe dry eye. Tear osmolarity values should be interpreted as an indicator of DED evolutionary process to severity.

Eye discomfort and air pollution.

Discomfort eye syndrome (DES) comprises a series of ‘minor’ subjective symptoms in patients where no relevant clinical signs are observed suggesting ocular disease. Our study includes 100 DES patients, excluding video terminal users, selected from the First Aid Service of our Department over two peak periods in both winter and summer time. The Schirmer test I, ferning test, breakup time and conjunctival cytology (scraping and imprint) were performed and data were related to sex, age and air pollution indexes, recorded in the patients’ living zones. Our results demonstrate that: (i) the ocular surface cytology and the analysis of tear film changes provide significant information in those patients where no other clinical signs are evident; (ii) DES symptoms are more frequent in women than in men (ratio about 2:1), both with ages over 51 years; (iii) DES is significantly associated with ocular surface inflammation, as detected by cytological methods, and (iv) ocular surface subclinical inflammation and ocular dryness are related to high concentrations of atmospheric polluters, in both sexes.

Tear proteomics in evaporative dry eye disease

Purpose:To analyze tear protein variations in patients suffering from dry eye symptoms in the presence of tear film instability but without epithelial defects.Methods:Five microlitres of non-stimulated tears from 60 patients, suffering from evaporative dry eye (EDE) with a break-up time (BUT) <10 s, and from 30 healthy subjects as control (no symptoms, BUT >10 s) were collected. Tear proteins were separated by mono and bi-dimensional SDS-PAGE electrophoresis and characterized by immunoblotting and enzymatic digestion. Digested peptides were analyzed by liquid chromatography coupled to electrospray ionization quadrupole-time of flight mass spectrometry followed by comparative data analysis into Swiss-Prot human protein database using Mascot. Statistical analysis were performed by applying a t-test for independent data and a Mann–Whitney test for unpaired data (P<0.05).Results:In EDE patients vscontrols, a significant decrease in levels of lactoferrin (data in %±SD): 20.15±2.64 vs24.56±3.46 (P=0.001), lipocalin-1: 14.98±2.70 vs17.73±2.96 (P=0.0001), and lipophilin A–C: 2.89±1.06 vs3.63±1.37 (P=0.006) was revealed, while a significant increase was observed for serum albumin: 9.45±1.87 vs3.46±1.87 (P=0.0001). No changes for lysozyme and zinc α-2 glycoprotein (P=0.07 and 0.7, respectively) were shown. Proteomic analysis showed a downregulation of lipophilin A and C and lipocalin-1 in patients, which is suggested to be associated with post-translational modifications.Conclusions:Data show that tear protein changes anticipate the onset of more extensive clinical signs in early stage dry eye disease.

Diagnostic performance of tear function tests in Sjogren's syndrome patients

ObjectiveTo evaluate the diagnostic performance of the tests included in primary Sjogrens syndrome (SS-I) diagnostic criteria (Schirmer I, break-up time, vital dye staining) and to compare them with other examinations related to the ocular surface status.MethodsClinical and cytological data were collected from 177 patients (62 SS-1, 56 non-SS autoimmune diseases, 59 Sicca syndrome). Tear tests included: a validated questionnaire on symptoms, Schirmer I, Jones test, Ferning test, BUT, corneal aesthesiometry, tear clearance test, lissamine green staining, impression conjunctival cytology. Data were statistically evaluated and sensitivity, specificity, likelihood ratio (LR+), receiver-operating characteristics (ROC) curves were calculated for each test.ResultsData showed a poor diagnostic performance of Schirmer test I (sensitivity 0.42; specificity 0.76; LR+1.75) and BUT (sensitivity 0.92; specificity 0.17; LR+1.11) (area under the curve in ROC analysis <0.58). Validated subjective symptoms questionnaire (sensitivity 0.89; specificity 0.72; LR+3.18), Jones test (sensitivity 0.60; specificity 0.88; LR+5), corneal aesthesiometry (sensitivity 0.80; specificity 0.67; LR+2.42), and tear clearance test (sensitivity 0.63; specificity 0.84; LR+3.93), all exhibited a high diagnostic performance (area under the curve in the ROC analysis always >0.70). Lissamine green staining exhibited the best performance (sensitivity 0.63; specificity 0.89; LR+5.72) but the result could be distorted by an incorporation bias.ConclusionsOur data suggest to implement the items for ocular signs and symptoms contained in many SS-I diagnostic criteria with the use of a validated questionnaire, performance of Jones test, corneal aesthesiometry measurement, and tear clearance rate evaluation.

Menopause and dry eye. A possible relationship

The hormonal status and in particular sex steroids play a role in the ocular surface homeostasis and function, during the whole life and in both sexes, accomplished by estrogenic and androgenic receptors located on corneal and conjunctival epithelia and Meibomian gland. The ocular surface is an integrated unit and any dysfunction results in a scarce or unstable preocular tear film which produces dry eye, a chronic inflammatory condition with increasing incidence as people get older but always more frequent among females. In post-menopausal women endocrine changes join the aging effects in the pathogensis of dry eye, but still it remains controversial whether estrogen or androgen deficiency or their imbalance impair ocular surface function. Another questionable issue concerns the efficacy of hormonal replacement therapy in the amelioration of dry eye symptoms and recovery of tear function, since the scientific literature stands in between a therapeutic or a promoting effect of eye dryness. Therapy of dry eye is usually based upon topical administration of tear substitutes; estrogen or androgen-based eye drops represents a promising innovative treatment based upon important scientific rationale.

The Biocompatibility of Silicone, Fluorosilicone and Perfluorocarbon Liquids as Vitreous Tamponades

Purpose: To investigate the interactions occurring at the interface between some currently used vitreous tamponading substances and the ocular tissues, in an early follow-up after surgery. Methods: Adult albino rabbit eyes underwent vitrectomy and were injected intravitreally with silicone oils at 1,000 or 3,000 cSt, fluorosilicone oil and perfluorodecalin. Different morphological techniques (light microscopy, scanning and electron microscopy, immunohistochemistry) were applied. Results: All the tested materials, although non-toxic, penetrate the ocular tissues also at the anterior segment level, and in the long-term follow-up this can yield functional impairment. No massive inflammation has been detected in the zones in contact with the materials, but immunoglobulins and complement fractions are anyway present in the stroma of various tissues and around the droplets of emulsified materials, suggesting a local immune reaction. Conclusions: Data from this study confirmed that a permanent vitreous substitute, showing a perfect biocompatibility, still does not exist. The indication to remove the tamponading substance within 2 months after the surgery emerges from this investigation, confirming previous studies. Despite some negative features, silicone oil still appears the most biocompatible material for vitreous replacement.

Hyperosmolar Stress Upregulates HLA-DR Expression in Human Conjunctival Epithelium in Dry Eye Patients and In Vitro Models

PURPOSE To investigate the immune response of human conjunctival epithelium to hyperosmolar stress. METHODS Tear osmolarity was measured in 15 normal subjects and 25 dry eye (DE) patients; conjunctival imprint cytology samples were obtained at the nasal bulbar area. Subconfluent primary human conjunctival epithelial cells (pHCECs) and human conjunctival organ cultures (hCOCs), both cultured in iso-osmolar medium (305 mOsm/L), were exposed for 24 hours to media with progressively higher osmolarity, with or without the ion channel inhibitor ruthenium red (RuR). Human leukocyte antigen (HLA)-DR expression was evaluated by immunocytochemistry, on imprints from subjects, on primary human conjunctival epithelial cells, on formalin fixed-paraffin embedded hCOCs, and by RT-PCR. Statistical evaluation was performed by applying the unpaired Students t test, as well as Spearmans rho and Pearsons r correlation coefficients (significance P < 0.05). RESULTS HLA-DR expression increased in DE subjects with respect to control (% mean ± SD, respectively, 46.16 ± 7.2 vs. 7.48 ± 1.14, P < 0.0001) and exhibited significantly high correlations with tear osmolarity values (r = 0.614; P < 0.0001). In vitro experiments showed a progressive increase in HLA-DR expression as the osmolarity of the medium was increased from 6.75 ± 1.16 (% mean ± SD) in iso-osmolar-cultured cells to 9.96 ± 1.37 and 12.94 ± 4.04 in cells cultured in, respectively, 350 and 400 mOsm/L (P < 0.05). A stepwise progressive increase was also found in hCOCs. Results were confirmed by RT-PCR. Ruthenium red significantly reduced HLA-DR expression in hyperosmolar-cultured cells. CONCLUSIONS Data from complementary techniques demonstrate that extracellular hyperosmolarity induces HLA-DR overexpression in human conjunctival epithelial cells in both DE patients and in vitro cell culture models.

Efficacy of standardized and quality-controlled cord blood serum eye drop therapy in the healing of severe corneal epithelial damage in dry eye.

Purpose: We standardized quality-controlled cord blood serum (CBS)–based eye drops and evaluated the efficacy of 1-month CBS treatment in the healing of diseased corneal epithelium in severe dry eye (DE) patients. Methods: Seventeen graft-versus-host disease (GVHD) and 13 Sjogren syndrome patients with severe persistent corneal defects were enrolled in the framework of a registered clinical trial (ClinicalTrials.gov NCT01234623). Sterile CBS eye drops were prepared to supply 0.15 ng per eye per day epithelial growth factor and administered for 1 month in a 1-day dose dispensing. The extent of epithelial defect was evaluated in square millimeters area, and subjective symptom score (Ocular Surface Disease Index score), Schirmer test I, break-up time, tear osmolarity, corneal esthesiometry (Cochet–Bonnet esthesiometer), conjunctival scraping, and imprint cytology with goblet cell count were performed at baseline (V0) and after 15 (V1) and 30 (V2, endpoint) days of treatment. Satisfaction and tolerability questionnaires were evaluated at V1 and V2. Results: A significant reduction was shown at the endpoint versus baseline in corneal epithelial damage (mean ± SD, 16.1 ± 13.7 vs. 40.9 ± 30 mm2/area, respectively), discomfort symptoms (Ocular Surface Disease Index score, 22.3 ± 10.3 vs. 39.3 ± 16.9), scraping cytology score (3.8 ± 1.2 vs. 6.6 ± 2.1), and tear osmolarity (312.5 ± 7 vs. 322 ± 9.1 mOsm/L), whereas a significant improvement was shown in corneal esthesiometry (48.2 ± 2.1 vs. 49.7 ± 2.1 nylon/mm/length, P < 0.05). All patients reported a high degree of satisfaction upon drop instillation. Conclusions: Heterologous CBS-based eye drops represent a promising therapeutic approach in the healing of severely injured corneal epithelium and in subjective symptom relief. These drops can be obtained as readily available and quality-controlled blood derivative from cord blood banks on a routine basis.

Dryness Symptoms, Diagnostic Protocol and Therapeutic Management: A Report on 1,200 Patients

Purpose: To report the diagnostic and therapeutic data obtained from 1,200 patients suffering from dry eye symptoms not due to Sjögren’s syndrome or other auto-immune diseases. Methods: Schirmer test I, ferning test, breakup time, vital dye staining, brush and imprint cytology were performed; data were grouped into diagnostic profiles, and the therapy was prescribed according to these. Results: Eight diagnostic profiles were identified. Dry eye was diagnosed in 57.1% of patients; the remaining 42.9% were found to suffer from eye discomfort or conjunctivitis of different aetiologies. Conclusions: Subjective symptoms of dryness can hide diseases other than dry eye; combined clinical and laboratory tests are requested to make a diagnosis. Our experience indicates that a therapy prescribed on the basis of diagnostic profiles provides relief in 79.1% of cases.

Detection of mucus glycoconjugates in human conjunctiva by using the lectin‐colloidal gold technique in TEM

Abstract The mucus glycoconjugates produced by conjunctival goblet cells in dry‐eye patients were studied by a specific cytochemical reaction in Transmission Electron Microscopy (TEM). Four lectins, proteins of vegetal origin which specifically bind glycosidic residues, (WGA, PNA, SBA and ConA) were used conjugated with colloidal gold as ultrastructural marker. We performed a quantitative analysis by counting the colloidal gold particles present on mucus granules. The results were compared with normal conditions. We found a decrease in sialic acid, N‐acetyl‐glucosamine, N‐acetyl‐galactosamine and galactose‐N‐acetyl‐galacto‐samine and an increase in mannose. The different content of glycoconjugates in goblet cells may reflect in the change of physical and functional properties of mucus. We think these data may be useful in the search for a therapeutic mucomimetic drug.