A protein dye-binding assay on cellulose membranes for tear protein quantification: Use of conventional Schirmer strips

PURPOSE: To develop a method to quantify tear protein concentration with the sensitivity to measure this variable in the restricted volumes of single human tear samples. METHODS: Aliquots of tear fluid from healthy subjects and a solution of standard bovine serum albumin (BSA) were spotted on cellulose membranes. Membranes were fixed, stained for protein with Coomassie blue, and washed until they displayed clear backgrounds. Stained spots were excised and eluted in a defined volume of methanol-ammonia, and the absorbance was determined spectrophotometrically at 610 nm. Membranes were calibrated by calculating their apparent thickness from the areas of stained spots and the corresponding aliquot volumes of either tear fluid or BSA solution. RESULTS: In our dye-binding assay, absorbance (0-1.00 OD) was found to have a linear relation with tear fluid volume (1-7 μL). In a study involving samples from 33 healthy subjects, aliquots (3 μL) of tear fluid were found to yield absorbances in th

Use of polyurethane minisponges to collect human tear fluid

Purpose: To characterize a method of tear collection based on the use of amphiphilic polyurethane absorbing minisponges. Methods: Tear fluid was collected from 17 healthy volunteers. A preweighed polyurethane dry minisponge was laid on the margin of the lower eyelid. Once wet (5-10 minutes), the fluid was transferred to a preweighed Eppendorf tube after squeezing the sponge by centrifugation. The amount of fluid absorbed and fluid recovered were determined by reweighing the sponge and the tube after absorption and centrifugation steps, respectively. The fluid was qualitatively characterized by electrophoretic polypeptide profiling in Coomassie blue-stained SDS-polyacrylamide gels. Results: Per eye, 14.6 ± 5.3 μL tear fluid was collected. That volume was about 90% of the fluid absorbed by polyurethane minisponges, almost doubling the fraction recovered from other more hydrophilic absorbing polymers. Major bands characterizing the electrophoretic profile of this fluid were those of 79,

Isolated conjunctival granuloma as a first manifestation of Parinaud's oculoglandular syndrome: A case report

Purpose: Parinaud's oculo-glandular syndrome (POGS) is the most frequent manifestation of ocular bartonellosis, and usually presents with local lymphadenopathies and systemic symptoms. We present a case of isolated conjunctival granuloma as the sole manifestation of ocular bartonellosis. Observations: A 67-year-old female presented to the authors’ eye clinic with complaints of a 2-week history of unilateral red eye and chemosis. Slit lamp examination revealed an isolated bulbar conjunctival granuloma. The remainder of the eye examination was unremarkable. Topical treatment with gatifloxacin and prednisolone acetate was started. Etiological work-up was performed. General laboratory tests revealed only a mild leukocytosis, and interferon gamma-release assay and chest computed tomography were normal. Serological testing for Bartonella henselae was positive at titers of 1:1024. Three weeks after initial symptoms, lymphadenopathies, malaise, and fever appeared. Systemic azithromycin was added, which resulted in complete regression of the disease. Conclusion and importance: Conjunctival granulomas present a wide range of differential diagnoses to the practitioner. Ocular bartonellosis is a relevant cause of conjunctival granuloma. POGS should be suspected in cases of conjunctival granulomata non-responsive to local therapy. It is important to consider that other agents to treat POGS have been described and are available, and that appropriate serological tests should be performed. Keywords: Ocular bartonellosis, Conjunctival granuloma, Parinaud, Cat scratch diseas

Progress in tear microdesiccate analysis by combining various transmitted-light microscope techniques

BACKGROUND: Tear desiccation on a glass surface followed by transmitted-light microscopy has served as diagnostic test for dry eye. Four distinctive morphological domains (zones I, II, III and transition band) have been recently recognized in tear microdesiccates. Physicochemical dissimilarities among those domains hamper comprehensive microscopic examination of tear microdesiccates. Optimal observation conditions of entire tear microdesiccates are now investigated. One-μ! aliquots of tear collected from individual healthy eyes were dried at ambient conditions on microscope slides. Tear microdesiccates were examined by combining low-magnification objective lenses with transmitted-light microscopy (brightfield, phase contrasts Ph1,2,3 and darkfield. RESULTS: Fern-like structures (zones II and III) were visible with all illumination methods excepting brightfield. Zone I was the microdesiccate domain displaying the most noticeable illumination-dependent variations, namely transparent band delimited by an outer rim (Ph1, Ph2), homogeneous compactly built structure (brightfield) or invisible domain (darkfield, Ph3). Intermediate positions of the condenser (BF/Ph1, Ph1/Ph2) showed a structured roughly cylindrical zone I. The transition band also varied from invisibility (brightfield) to a well-defined domain comprising interwoven filamentous elements (phase contrasts, darkfield. CONCLUSIONS: Imaging of entire tear microdesiccates by transmitted-light microscopy depends upon illumination. A more comprehensive description of tear microdesiccates can be achieved by combining illumination methods

Microdesiccates produced from normal human tears display four distinctive morphological components

Desiccation of human tears on glass surfaces results in fern-like crystalloids. This phenomenon has been associated with tear normality (Tear Ferning Test, TFT) and is used as a diagnostic aid to evaluate patients with Dry-Eye disease. However, TFT is focused on the assessment of only a minor fraction of desiccated tear samples and considers only the relative abundance and density of fern-like crystalloids. The aim of this study was to characterize morphologically entire desiccated micro volumes of tears from healthy donors. Tear samples were collected from 23 healthy young adult volunteers. Tear aliquots (1-3 μL) were allowed to dry on glass surfaces under ambient conditions of temperature (15-25°C) and relative humidity (40-45%). Dry samples were analyzed by dark-field microscopy. Morphometric data were acquired with Image J software. Tear volume was positively correlated with both area and time of desiccation. Morphological features of multiple microdesiccates produced from a single subject displayed striking similarities whereas tear microdesiccates from different healthy subjects displayed consistent differences but shared a common general design. This design may be mostly represented by the occurrence of four distinctive zones, named as zones I, II, III and Transition band. The main features of these zones are described

Zone i of Tear Microdesiccates Is a Lipid-Containing Structure

Copyright © 2016 Wolters Kluwer Health, Inc. All rights reserved. Purpose: Morphological features of tear microdesiccates on glass surfaces have been associated with tear fluid status. Tear-film lipids play a critical role in the pathophysiology of some ocular surface disorders. Tear microdesiccates display 4 distinctive morphological domains (zones I, II, III, and transition band). In this study, we investigated the lipid location in tear microdesiccates. Methods: Tear from individual healthy eyes (assessed by symptoms, signs, and slit-lamp examination) was collected using absorbing minisponges. One-μL aliquots were allowed to dry under ambient conditions on microscope slides. Tear microdesiccates were examined by various transmitted light microscopy methods. Tear lipids were located both by partition experiments using 2 lipophilic dyes (Oil red O and Nile blue A) mixed with tear fluid under conditions preserving morphological features of microdesiccates and by assessing the effect o

Accelerated collagen cross-linking in the management of advanced Acanthamoeba keratitis

ABSTRACT Purpose: To report our initial experience in the treatment of Acanthamoeba keratitis with accelerated corneal collagen cross-linking. Methods: Retrospective chart review of patients diagnosed with Acanthamoeba keratitis with progressive corneal melting who were treated with accelerated collagen cross-linking. Results: A total of 6 eyes (5 patients) were reviewed. All the patients received adjuvant therapy with moxifloxacin and chlorhexidine. In 4 cases, the ulcer healed with a mean interval to epithelialization of 108.8 days (range 59-217). In 2 eyes, there was a persistent neurotrophic ulcer. The melting was not progressive in any case, nor did any eye required emergency penetrating keratoplasy. Conclusion: This study suggests a beneficial effect of accelerated collagen cross-linking in cases of Acanthamoeba keratitis with corneal melting. Thus, collagen cross-linking may be considered as adjuvant treatment for Acanthamoeba keratitis

Conjunctival impression cytology in patients with normal and impaired OSDI scores Citología de impresión conjuntival en pacientes con valores OSDI normales y alterados

© 2013 Sociedad Española de Oftalmología. Published by Elsevier España, S.L.U. All rights reserved. Purpose: To describe goblet cell density and Nelson grading in different areas of the ocu-lar surface using conjunctival impression cytology (CIC) among patients with normal andimpaired Ocular Surface Disease Index (OSDI) scores. Material and methods: Patients (n = 166) under assessment for dry eye were recruited between 2011 and 2012 and classified according to the OSDI score in 4 categories (normal and impaired). Cytological study (CIC plus Papanicolaou staining) using the Nelson grading system, with modifications in staging, and goblet cell counting were performed on the nasal, temporal, inferior, and superior bulbar conjunctival surfaces. Results: Nelson grading was significantly higher in patients with a severely impaired OSDI score (1.41 ± 0.14) compared to normal patients (0.86 ± 0.09) (P<.01). Goblet cell density was significantly reduced in patients with a severely impaired OSD

Algorithm approach for revision surgery following late-onset bleb complications after trabeculectomy: Long-term follow-up Algoritmo de abordagem para cirurgia de revisão nas complicações da bolha de início tardio após trabeculectomia: Acompanhamento de lo

Purpose: The aim of this study was to introduce a reproducible algorithm for the surgical management of late-onset (>2 months) bleb complications after trabeculectomy with mitomycin C. Methods: We performed a retrospective review of eyes treated using a reproducible algorithm approach by a single surgeon for the surgical management of late-onset bleb complications from July 2006 to April 2014. Exclusion criteria were bleb revision with less than 3 months of follow-up or bleb revision combined with other glaucoma procedures at the time of surgery. Success was evaluated using the Kaplan-Meier survival method and defined as achieving all of the following criteria: Primary surgery indication resolved, no additional surgery required for decreasing the intraocular pressure (IOP), and IOP of >6 mmHg and <18 mmHg. Results: Twenty-three eyes from 20 patients were evaluated. Indications for bleb revision were hypotonic maculopathy (47.8%), bleb leak (30.4%), and dysesthetic bleb (21.7%). The ov