Expanded biochemical analyses of human tear fluid: Polyvalent faces of the schirmer strip.

The tear film forms a protective barrier between the ocular surface and the external environment. Despite its small volume, recent advancements in preanalytical and analytical procedures have enabled its in-depth analysis using multiple approaches. However, the diversity of tear film collection methods and the lack of standardization in pre-analytical methods represent the main obstacles to reproducible results and comparison among different studies. In this study, we first improved the pre-analytical procedures for the extraction of various molecular entities from Schirmer strips (ScS). Subsequently, our investigation focused on analyzing the molecular variances that might occur between two primary tear collection methods: capillary tube (CT) and ScS. Additionally, we examined different parts of the ScS to underscore these variations, which could serve as crucial factors for developing a standardized, optimized protocol for sample processing. Our results show that the inclusion of surfactants in the extraction process enhanced both the yield of protein extraction and the number of proteins identified in ScS, by effectively lysing the cells and improving the solubility of several intracellular proteins. In addition to proteins, nucleic acids could also be recovered for gene expression analyses, particularly from the bulb region of the ScS which is placed in the cul-de-sac. Despite their diluted nature, extracts from ScS remain a suitable material for retrieving tear proteins such as IL-17A at levels as low as the fg/mL range, thanks to highly sensitive immunoassays. Collection methods can affect measured tear protein levels. Lactoferrin is found in higher percentages in capillary electrophoresis analysis of tears collected using ScS compared to tears collected by CT (39.6 ± 4.8% versus 31 ± 4.4%).

In vivo confocal microscopic study of corneal innervation in Sjögren's Syndrome with or without small fiber neuropathy.

PURPOSE: To study changes in the subbasal nerve plexus by In vivo confocal microscopy (IVCM) in Sjögren's Syndrome (SS) with or without associated Small Fiber Neuropathy (SFN), in order to prevent diagnostic delay. METHODS: Seventy-one patients with SS, including 19 with associated SFN, 20 healthy volunteers and 20 patients with Meibomian gland dysfunction (MGD) were included in this retrospective case-control study. IVCM was used to investigate subbasal nerve plexus density and morphology. RESULTS: Corneal sensitivity as evaluated with the Cochet-Bonnet aesthesiometer was significantly reduced in the SS group versus the control group (P = 0.026) and the MGD group (P = 0.037). The number of inflammatory cells was significantly increased in the SS group to 86.2 ± 82.1 cells/mm2 compared to the control group (P < 0.001). The density of the subbasal nerve plexus was significantly reduced to 16.7 ± 6.5 mm/mm2 in the SS group compared to the control group (P < 0.005) and the MGD group (P = 0.042). The tortuosity of the nerves in the SS group was significantly increased compared to the control group (P < 0.001) and the MGD group (P = 0.025). The average number of subbasal nerve plexus neuromas was significantly increased in the SS group compared to the control group (P = 0.001), with a significant increase in the average number of neuromas in SS patients with associated SFN compared to SS patients without SFN (P = 0.008). CONCLUSION: IVCM can be useful to detect corneal nerve changes in SS patients and may allow earlier diagnosis of the disease and to consider new therapeutic approaches.

Assessment of patient burden from dry eye disease using a combination of five visual analogue scales and a radar graph: a pilot study of the PENTASCORE.

BACKGROUND/AIMS: Dry eye disease (DED) questionnaires would ideally be easy and fast to answer and explore the main aspects of disease burden and satisfaction (efficacy and tolerability) with treatment. This pilot study evaluates the Pentascore questionnaire for routinely assessing DED. METHODS: The Pentascore combines five visual analogue scales (VAS) to assess the intensity and frequency of ocular pain/discomfort, the impact of DED on daily activities and visual tasks and the efficacy and tolerability of ongoing DED treatment(s). This retrospective study compared Pentascore to the Ocular Surface Disease Index (OSDI) questionnaire, fluorescein tear break-up-time, corneal staining and Schirmer I test. RESULTS: For 161 DED patients, the algebraic mean (±SE) for the Pentascore was 52.6±1.8, the mean standardised area of the radar graph was 32.1±1.7 (out of 100) and the mean score for the OSDI was 52.6±1.8. Both questionnaires were highly statistically correlated (R=0.74 for both algebraic score and radar area, p<0.001), and each of five Pentascore VAS was significantly correlated with the OSDI (p<0.05). Corneal staining score (CSS) was correlated with two Pentascore VAS (impact of DED on daily activities and visual tasks), and there was a trend towards a correlation between CSS and the area of the radar graph (p=0.09). CONCLUSIONS: This pilot study indicates that the Pentascore can rapidly and effectively assess the burden of DED and satisfaction with treatments. Compared with the algebraic mean, the estimation of the area of the radar graph likely improves the sensitivity for detecting differences/changes in symptoms and treatment follow-up.

Corneal Nerve Abnormalities in Painful Dry Eye Disease Patients.

Background: This study aimed to compare the corneal nerve structural abnormalities detected using in vivo confocal microscopy (IVCM) in patients with neuropathic corneal pain (NCP) secondary to primary meibomian gland dysfunction (MGD) or autoimmune dry eye (AIDE). Methods: A two-stage retrospective nested case-control study was conducted. First, data from patients with either MGD or AIDE were assessed, selecting only cases with no corneal pain (VAS = 0) or severe pain (VAS ≥ 8). Ocular signs and symptoms of the 238 selected patients were compared between painful and painless cases. Next, painful patients with no corneal damage (Oxford score ≤ 1) were selected within each study group, defining the cases with NCP (i.e., "pain without stain"). IVCM images from all groups were compared with prospectively-recruited healthy controls, focusing on dendritiform cell density and nerve abnormalities (density, tortuosity, microneuromas). Results: AIDE patients had more ocular signs/symptoms than MGD patients. Compared with healthy controls, AIDE-related NCP patients showed increased nerve tortuosity and number of neuromas, whereas MGD-related NCP patients had reduced nerve density and increased number, perimeter, and area of microneuromas. Microneuromas were also observed in healthy controls. Furthermore, a higher number of microneuromas was found in MGD-related NCP compared to AIDE-related NCP or painless MGD. Conclusions: MGD-related NCP was associated with significantly more corneal nerve abnormalities than AIDE-related NCP or healthy controls. Although IVCM can be useful to detect NCP-related corneal nerve changes in such patients, the diagnosis of dry eye disease-related NCP will require an association of several IVCM-based criteria without relying solely on the presence of microneuromas.

Assessment of corneal epithelial thickness mapping in epithelial basement membrane dystrophy.

PURPOSE: To investigate the corneal epithelial thickness topography with optical coherence tomography (OCT) and its relationship with vision quality in epithelial basement membrane dystrophy (EBMD). METHODS: 45 eyes of EBMD patients, 26 eyes of dry eye (DED) patients and 22 eyes of normal subjects were enrolled. All participants were subjected to 9-mm corneal epithelial mapping with OCT and vision quality was assessed with the optical quality analysis system using the objective scatter index (OSI). Central, superior, inferior, minimum, maximum, and standard deviation of epithelium thickness (Irregularity), were analysed and correlations with the OSI were calculated. RESULTS: The mean (±SD) central, inferior and maximum epithelial thicknesses of the EBMD patients (respectively, 56.4 (±8.1) µm, 58.9 (±6.4) µm, and 67.1 (±8.3) µm) were thicker compared to DED patients (P<0.05) and normal subjects (P<0.05). We found greater irregularity of epithelial thickness in EBMD (5.1±2.5 µm) compared to DED patients (2.6±1.0 µm) (P = 4.4.10-6) and normal subjects (2.1±0.7 µm) (P = 7.6.10-7). The mean OSI was worse in EBMD patients than in DED patients (P = 0.01) and compared to normal subjects (P = 0.02). The OSI correlated with the epithelial thickness irregularity (Spearman coefficient = 0.54; P = 2.65.10-5). CONCLUSIONS: The OCT pachymetry map demonstrated that EBMD patients had thicker corneal epithelium in the central and inferior region. These changes were correlated with objective measurements of vision quality. This OCT characterisation of the EMBD provides a better understanding of the epithelial behaviour in this dystrophy and its role in vision quality.

The Role of Meibography in the Diagnosis of Meibomian Gland Dysfunction in Ocular Surface Diseases.

PURPOSE: To evaluate dysfunction in various ocular surface diseases (OSDs) including primary meibomian gland disease (MGD), perennial allergic conjunctivitis, and primary and secondary Sjögren syndromes. METHODS: A retrospective analysis of 146 patients (111 women and 35 men) with symptomatic OSDs was performed. Patients were divided into two groups: the non-MGD group (55 patients) and the MGD group (91 patients). All patients had an evaluation of ocular surface symptoms and clinical tests, including tear film breakup time (BUT), the first and the mean noninvasive breakup time (NIKBUTf and NIKBUTavg, respectively). The meibomian gland loss of the lower eyelid was quantified using meibography and the meiboscale. RESULTS: There was no significant difference regarding age or sex ratio between the two groups. The meiboscale in the MGD group was significantly higher than that in the non-MGD group (P = 0.003). The non-MGD patients were more symptomatic than those in the MGD group (P = 0.043). There were no significant differences between MGD and non-MGD groups regarding a Schirmer test (P = 0.195), BUT (P = 0.719), NIKBUTf (P = 0.96), or NIKBUTavg (P = 0.70). In the whole population, there was a negative correlation between meiboscale and NIKBUT (r = -0.21, P = 0.02), but no other correlations were found. CONCLUSIONS: Meibomian gland dysfunction was observed among different OSDs. Meibomian gland loss evaluated by meibography might help identify MGD in patients suffering from OSD. TRANSLATION RELEVANCE: Meibography provides a better understanding of MGD in several OSD. It may be useful to integrate this objective analysis to improve treatments of OSD associated to MGD.

Conjunctival Inflammatory Gene Expression Profiling in Dry Eye Disease: Correlations With HLA-DRA and HLA-DRB1.

Purpose: In several multicenter clinical trials, HLA-DR was found to be a potential biomarker of dry eye disease (DED)'s severity and prognosis. Given the fact that HLA-DR receptor is a heterodimer consisting in an alpha and a beta chain, we intended to investigate the correlation of inflammatory targets with the corresponding transcripts, HLA-DRA and HLA-DRB1, to characterize specific targets closely related to HLA-DR expressed in conjunctival cells from patients suffering from DED of various etiologies. Methods: A prospective study was conducted in 88 patients with different forms of DED. Ocular symptom scores, ocular-staining grades, tear breakup time (TBUT) and Schirmer test were evaluated. Superficial conjunctival cells were collected by impression cytology and total RNAs were extracted for analyses using the new NanoString® nCounter technology based on an inflammatory human code set containing 249 inflammatory genes. Results: Two hundred transcripts were reliably detected in conjunctival specimens at various levels ranging from 1 to 222,546 RNA copies. Overall, from the 88 samples, 21 target genes showed a highly significant correlation (R > 0.8) with HLA-DRA and HLA-DRB1, HLA-DRA and B1 presenting the highest correlation (R = 0.9). These selected targets belonged to eight family groups, namely interferon and interferon-stimulated genes, tumor necrosis factor superfamily and related factors, Toll-like receptors and related factors, complement system factors, chemokines/cytokines, the RIPK enzyme family, and transduction signals such as the STAT and MAPK families. Conclusions: We have identified a profile of 21 transcripts correlated with HLA-DR expression, suggesting closely regulated signaling pathways and possible direct or indirect interactions between them. The NanoString® nCounter technology in conjunctival imprints could constitute a reliable tool in the future for wider screening of inflammatory biomarkers in DED, usable in very small samples. Broader combinations of biomarkers associated with HLA-DR could be analyzed to develop new diagnostic approaches, identify tighter pathophysiological gene signatures and personalize DED therapies more efficiently.

Impact of Dry Eye Disease on Vision Quality: An Optical Quality Analysis System Study.

PURPOSE: We evaluated the relationship between ocular surface clinical tests and quality of vision in patients with dry eye disease (DED). METHODS: In this study, 136 eyes of 72 dry eye patients were evaluated retrospectively using the ocular surface disease index (OSDI), measurement of tear film break-up time (TBUT), the Oxford score, Van Bijsterveld score, and Schirmer I test. Quality of vision was assessed with the optical quality analysis system (OQAS) using the objective scatter index (OSI) recorded over 20 seconds without blinking. Correlations between dry eye symptoms and signs, and OSI measurements were evaluated. RESULTS: The OSI and OSI standard deviation (OSI SD) were correlated with TBUT (r = -0.21, P = 0.013 and r = -0.18, P = 0.038, respectively), Oxford score (r = 0.31, P = 0.0002 and r = 0.18, P = 0.032, respectively), and the Van Bijsterveld score (r = 0.33, P = 0.0001 and r = 0.25, P = 0.003, respectively). The OSI also was correlated with the Schirmer test (r = -0.19, P = 0.025), OSDI (r = 0.17, P = 0.04), and the ocular symptoms subscale of the OSDI (r = 0.21, P = 0.01). OSI SD was correlated with the environmental triggers subscale of the OSDI (r = 0.21, P = 0.016). CONCLUSIONS: Quality of vision measured with the OQAS was correlated with dry eye symptoms and signs. The OQAS could be a useful tool to better evaluate visual function in patients with DED. TRANSLATIONAL RELEVANCE: The OQAS provides a better understanding of patient complaints about alteration of vision quality. It might be useful to integrate this objective system in severity assessments and follow-up of DED, especially for treatment evaluations.

Proinflammatory Markers, Chemokines, and Enkephalin in Patients Suffering from Dry Eye Disease.

Dry eye symptoms are among the leading complaints in ophthalmology. Dry eye disease (DED) is associated with significant pain affecting quality of life. Cellular and molecular mechanisms underlying ocular pain associated with DED are not fully understood. In this study, we investigated the ocular surface of patients with DED using in vivo confocal microscopy (IVCM) to quantify corneal nerve density and its relation with corneal inflammation. Gene expression of the proinflammatory markers HLA-DR, IL-6, CXCL12, and CCL2 and the receptors CXCR4 and CCR2, as well as PENK (enkephalin precursor), was therefore quantified in conjunctival impression cytology specimens. Thirty-two patients with DED and 15 age-matched controls were included. Subbasal nerve density was significantly lower in DED patients compared to controls. IVCM analysis revealed that DED patients had a significantly higher corneal dendritic cell density compared to controls. Conjunctival impression cytology analysis revealed that HLA-DR, IL-6, CXCR4, and CCL2/CCR2 mRNA levels were significantly increased in DED patients compared to controls, whereas PENK mRNA levels were significantly decreased. Similar results were obtained in vitro on immortalized human conjunctiva-derived epithelial cells challenged with osmotic stress that mimics the DED condition. These results demonstrate that proinflammatory molecules and endogenous enkephalin have opposite gene regulation during DED.

The impact of dry eye disease on visual performance while driving.

PURPOSE: A specific simulator was used to assess the driving visual performance in patients with dry eye disease (DED) and to determine clinical predictors of visual impairments while driving. DESIGN: Prospective case-control study. METHODS: The study was conducted in the Center for Clinical Investigation of Quinze-Vingts National Ophthalmology Hospital, Paris, France. Twenty dry eye patients and 20 age- and sex-matched control subjects were included. Vision-related driving ability was assessed using a specific driving simulator displaying randomly located targets with a progressive increase in contrast to be identified. Other examinations included clinical examinations, serial measurements of corneal higher-order aberrations (HOAs), and vision-related quality-of-life questionnaire (Ocular Surface Disease Index [OSDI]). Data collected during driving test (ie, the number of targets seen, their position, and the response time) were compared between groups and analyzed according to clinical data, aberration dynamics, and quality-of-life index. RESULTS: The percentage of targets missed as well as average response time were significantly increased in DED patients as compared with controls (P < .01). More specifically, the visual function of DED patients was more impaired in specific situations, such as crossroad or roundabout approaches. In DED patients, the response time was found to positively correlate with the progression index for HOAs (P < .01) and with the OSDI "symptoms" subscale (P < .05). CONCLUSIONS: Degradation of ocular optical qualities related to DED is associated with visual impairments during driving. This study objectively has demonstrated the impact of tear film-related aberration changes on activities of daily living in DED.

Tear film aberration dynamics and vision-related quality of life in patients with dry eye disease.

OBJECTIVE: Corneal and ocular wavefront aberrations were recorded together with clinical examination results and patient-reported vision-related quality-of-life evaluation results to define the relevance of dynamic optical analysis of the eye in dry eye disease (DED). DESIGN: Prospective and comparative clinical study. PARTICIPANTS: Forty DED patients and 40 age- and gender-matched control subjects. METHODS: Serial measurements of ocular and corneal higher-order aberrations (HOAs) after blink were performed for 10 seconds using the KR-1 aberrometer (Topcon, Clichy, France). Vision-related health-targeted quality of life was evaluated using the Ocular Surface Disease Index (OSDI) questionnaire. The clinical examination included tear film assessment (tear film break-up time and Schirmer I test), ocular surface damage assessment with the Oxford and van Bijsterveld indexes, and Meibomian dysfunction grading. Tear osmolarity also was measured. MAIN OUTCOME MEASURES: The time course of HOAs and modulation transfer function (MTF) was compared between groups and was analyzed in comparison with the OSDI and clinical data in DED patients. RESULTS: The root mean square of ocular and corneal total HOAs, particularly third-order aberrations, significantly increased over the 10-second period in DED patients, whereas no change occurred in controls. Analysis of MTF revealed progressive degradation of ocular optical quality resulting from loss of contrast at intermediate and high spatial frequencies in DED patients compared with controls. The progression index for corneal HOAs was correlated with the subjective index of patient-reported visual outcomes and with objective clinical findings of tear film and ocular surface damage. CONCLUSIONS: Objective measurement of the time course of HOAs may constitute a new single instrument to evaluate and manage patients with DED because it reliably reflects the completeness of the disease.