Human tear film protein sampling using soft contact lenses.

BACKGROUND: Human tear protein biomarkers are useful for detecting ocular and systemic diseases. Unfortunately, existing tear film sampling methods (Schirmer strip; SS and microcapillary tube; MCT) have significant drawbacks, such as pain, risk of injury, sampling difficulty, and proteomic disparities between methods. Here, we present an alternative tear protein sampling method using soft contact lenses (SCLs). RESULTS: We optimized the SCL protein sampling in vitro and performed in vivo studies in 6 subjects. Using Etafilcon A SCLs and 4M guanidine-HCl for protein removal, we sampled an average of 60 ± 31 µg of protein per eye. We also performed objective and subjective assessments of all sampling methods. Signs of irritation post-sampling were observed with SS but not with MCT and SCLs. Proteomic analysis by mass spectrometry (MS) revealed that all sampling methods resulted in the detection of abundant tear proteins. However, smaller subsets of unique and shared proteins were identified, particularly for SS and MCT. Additionally, there was no significant intrasubject variation between MCT and SCL sampling. CONCLUSIONS: These experiments demonstrate that SCLs are an accessible tear-sampling method with the potential to surpass current methods in sampling basal tears.

Tear Samples for Protein Extraction: Comparative Analysis of Schirmer's Test Strip and Microcapillary Tube Methods.

INTRODUCTION: Tear sampling is an attractive option for collecting biological samples in ophthalmology clinics, as it offers a non-invasive alternative to other invasive techniques. However, there are many tear sampling methods still in consideration. This study explores the suitability of Schirmer's test strip and microcapillary tube as reliable and satisfactory methods for tear sampling. METHODS: Tear samples were collected from eight healthy volunteers using the standard Schirmer's test strip method with or without anesthesia and microcapillary tubes. The total tear protein concentrations were analyzed via spectrophotometry and bicinchoninic acid (BCA) protein assay. The protein profile was determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal wetting length of Schirmer's strip and suitable buffer solutions were compared. Discomfort levels reported by participants and the ease of execution for ophthalmologists were also evaluated. RESULTS: Tear samples exhibited typical protein profiles as shown by SDS-PAGE. The mean total protein obtained from an optimum wetting length of 20 mm using Schirmer's strip without anesthesia in phosphate-buffered saline (PBS) yielded substantial quantities of protein as measured by nanophotometer (220.20 ± 67.43 µg) and the BCA protein assay (210.34 ± 59.46 µg). This method collected a significantly higher quantity of protein compared to the microcapillary tube method (p=0.004) which was much more difficult to standardize. The clinician found it harder to utilize microcapillary tubes, while participants experienced higher insecurity and less discomfort with the microcapillary tube method. PBS used during the tear protein extraction process eluted higher tear protein concentration than ammonium bicarbonate, although the difference was not statistically significant. Using anaesthesia did not ease the sampling procedure substantially and protein quantity was maintained. CONCLUSION: Good quality and quantity of protein from tear samples were extracted with the optimized procedure. Schirmer's strip test in the absence of local anesthesia provided a standard, convenient, and non-invasive method for tear collection.

SARS-CoV-2 and the Eye: A Relationship for a Possible Prognostic Tool in COVID-19 Patients.

PURPOSE: In December 2019 there was the first report about a new viral infection in Wuhan, China. The new virus was taxonomically designed as Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) causing the coronavirus disease 2019 (COVID-19). SARS-CoV-2 uses the angiotensin-converting enzyme 2 (ACE2) receptor for cell invasion, which is expressed in different tissues including lungs, small intestine, testicles, kidneys, brain, and the eye. The purpose of this article is to review the available information on the relationship of COVID-19 with the eye, as well as evaluating the possible usefulness of ocular diagnostic tests to help in the diagnosis and/or monitoring of patients with this disease. METHODS: We performed a retrospective review of relevant articles from November 2019 to April 2020. RESULTS: Ocular infection by SARS-CoV-2 is still controversial; nevertheless, the possibility of being a viral reservoir has been suggested, increasing the likelihood of infection. Some reports demonstrated the presence of SARS-CoV-2 in tears, and previously published data suggest a pathological increase of cytokine concentrations in COVID-19 patients; the cytokine release syndrome or cytokine storm contributes to lung and central nervous system damage. The usefulness of tears for the measurement of inflammatory cytokines in various diseases is well known, in particular IL-6, which has been correlated to the severity of COVID-19. CONCLUSION: Considering that the IL-6 signaling cascade may be activated in patients with COVID-19, makes it an excellent target for diagnostic and/or monitoring purposes.

Tear fluid collection methods: Review of current techniques.

Tear fluid, composed of lipid, aqueous, and mucin layers, contains electrolytes, water, proteins, peptides, and glycoproteins. Its components may serve as diagnostic indicators of local and systemic diseases. The aim of the study was to conduct literature review in order to identify the current methods of tear collection. The most commonly used method which was relatively easy to perform and allowed to obtain sufficient tear volume for further chemical and physical analysis was selected through PubMed database search for the following keywords: tear sampling, human tears, chemical analysis of tears, physical tear analysis, animal tear sampling. Final criteria of articles selection were: human tears, tear sample collection, chemical and physical analysis of tears. Time of publication of the articles not older than 1995. The analysis of 70 articles revealed that the most common tear fluid collection methods are Schirmer tear strips and capillary tubes. Thus, we recommend the use of Schirmer strips and microcapillary tubes as the cheapest and easiest methods for sampling of tear fluid for further chemical analysis.

Direct Measurement of a Biomarker's Native Optimal Frequency with Physical Adsorption Based Immobilization.

The optimal frequency (OF) of a biomarker in electrochemical impedance spectroscopy (EIS) is the frequency at which the EIS response best reflects the binding of the biomarker to its molecular recognition element. Commonly, biosensors rely on complicated immobilization chemistry to attach biological molecules to the sensor surface, making the direct study of a biomarker's native OF a challenge. Physical adsorption presents a simple immobilization strategy to study the native biomarker's OF, but its utility is often discouraged due to a loss in biological activity. To directly study a biomarker's native OF and investigate the potential of OF to overcome the limitations of physical adsorption, a combination of EIS and glutaraldehyde-mediated physical adsorption was explored. The experimental sensing platform was prepared by immobilizing either anti-lactoferrin (Lfn) IgG or anti-immunoglobulin E (IgE) onto screen printed carbon electrodes. After characterizing the native OFs of both biomarkers, investigation of the platform's specificity, stability, and performance in complex medium was found to be sufficient. Finally, a paper-based tear sampling component was integrated to transform the testing platform into a prototypical point-of-care dry eye diagnostic. The investigation of native OFs revealed a correlation between the native OFs (57.44 and 371.1 Hz for Lfn and IgE, respectively) and the molecular weight of the antibody-antigen complex. Impedance responses at the native OFs have enabled detection limits of 0.05 mg/mL and 40 ng/mL for Lfn and IgE, respectively, covering the clinically relevant ranges. The native OFs were found to be robust across various testing mediums and conditions.

The potential influence of Schirmer strip variables on dry eye disease characterisation, and on tear collection and analysis.

PURPOSE: The use of the Schirmer strips (SS) as a tool in the characterisation of dry eye disease, depends upon the quantitative assessment of tear production and constituents. The aim of this study was to ascertain the extent to which the properties of commercially available SS can vary and the way in which this baseline information may relate to their comparability in clinical use. METHODS: Five SS were analysed: Clement Clarke®, TearFlo®, Bio Schirmer®, Omni Schirmer® and JingMing®. Various aspects of their physical appearance and physicochemical behaviour were measured, including size, weight, and thickness together with surface morphology (assessed by SEM) and aqueous uptake and release behaviour (including the influence of each strip on protein retention and eluent osmolarity). RESULTS: All physical parameters varied between the strips studied for example the Clement Clark was the largest, thickest, and heaviest strip assessed in this study. SEM images showed that each of the SS had unique surface morphologies. Statistically significant differences among the strips were found for uptake (p=0.001) and release volume (p=0.014). Clement Clarke absorbed the highest volume over a fixed time period (23.8±1.6µl) and Omni the lowest (19.3±0.5µl). Clement Clarke showing the highest eluent osmolarity value (5.0±0.0mOsm/L) and TearFlo the lowest (2.8±0.4mOsm/L). CONCLUSION: The five strips investigated in this study indicate that there is no standardisation of commercial strips, despite the fact that the need for standardisation was recognised over fifty years ago. This study provides useful baseline information relating to SS comparability in clinical use.

Evaluation of commonly used tear sampling methods and their relevance in subsequent biochemical analysis.

The human precorneal tear film is a special body fluid, since it is a complex mixture of proteins, lipids, small bioactive molecules, and their concentrations and relative distribution represent not only the metabolic state of the ocular surface but also the systemic and local homeostasis of the outer eye and the human body. This suggests that biochemical analysis of the precorneal tear film composition may provide a non-invasive tool for diagnosis and monitoring of disease progression or treatment efficacy in human medicine. However, collecting tears is demanding, and obtaining reproducible and unaltered samples is challenging because of the small sample volumes of tears. Several methods are available for tear collection as a preparatory step of precorneal tear film analysis, and the collection method used has to be assessed since it has a critical impact on the effectiveness of the assays and on the quality of the results. Each sampling method has advantages and disadvantages; therefore, it is not easy to choose the appropriate collecting method for tear collection. To overcome these limitations various methods have been recommended by different authors for special aspects of specific tests. The aim of our review was to evaluate tear sampling methods with regard to our ongoing biochemical analysis. *Contributed equally.

Comparison of the Effectiveness between Sampling Methods for Protein Analysis of Tear Fluids

PURPOSE: Although various sampling methods of tears from conjunctival sac have been reported, no previous study compared their effectiveness or efficiency based on protein extraction. By comparing the compliance, volume and protein concentration of each tear sampling method, we searched for the most efficient tear collection method. METHODS: Resting tear samples of 14 eyes of normal subjects were collected using Schirmer paper, capillary tube, cellolose acetate rod and 3 different ophthalmic sponges made of different materials and density (Merocel(R), KeraCel(R) and Weck-Cel(R)). After centrifugation of the collected tear samples, the tear volume and protein concentration were measured for each method. Additionally, the compliance of each tear sampling method was analyzed by numerically representing the amount of discomfort experienced during resting tear collection. RESULTS: The average volume retrieved by each tear sampling method was 9.0 +/- 1.1 microL with no significant differences between groups. The average concentration of protein retrieved by each tear sampling method was 5.3 +/- 1.2 microg/microL. Merocel(R) retrieved 7.6 +/- 0.61 microg/microL, which was significantly higher than other sampling methods (p < 0.05). The compliance of Merocel(R) and the capillary tube were the highest, while KeraCel(R) showed the lowest compliance. CONCLUSIONS: Merocel(R) retrieved the highest amount of protein and showed high compliance and may be the most effective and easily applicable tear sampling method in clinical settings.

The fall and rise of tear albumin levels: a multifactorial phenomenon.

Albumin in tears is used as a diagnostic marker of ocular insult and inflammation, but whether its presence in tears is responsive or part of an adaptive reaction remains unresolved. A review of the literature on tear albumin concentration emphasizes that variables such as collection method, stimulus, assay technique, and disease state influence the quoted values to different extents. Influence of assay technique is negligible in comparison to variation in sampling conditions. Ocular disease increases albumin concentrations but not in a specific manner. The literature review also highlighted that little systematic research has been carried out on the daily cycle of tear albumin levels. In order to remedy this shortcoming, we investigated variations in tear albumin concentration during the waking day. The concentration of albumin in 400 tear samples collected from 13 subjects was assessed at 2-hourly intervals throughout the waking day. Highest daytime albumin concentrations were obtained within 10 minutes of waking, with a mean concentration of >50 ± 22 µg/ml. Albumin levels were at their lowest, but most consistent, 2-6 hours post-waking. This pattern was followed by a progressive increase in albumin concentration during the latter part of the day. Although individual subject-to-subject concentration differences were observed, this distinctive pattern of diurnal variation was found in all subjects. The results presented suggest a regulated, not random, pattern of variation within the period of study.

Comparison of two methods of tear sampling for protein quantification by Bradford method / Comparação de dois métodos de coleta de lágrima para quantificação proteica pelo método de Bradford

The aim of this study was to compare two methods of tear sampling for protein quantification. Tear samples were collected from 29 healthy dogs (58 eyes) using Schirmer tear test (STT) strip and microcapillary tubes. The samples were frozen at -80ºC and analyzed by the Bradford method. Results were analyzed by Student's t test. The average protein concentration and standard deviation from tears collected with microcapillary tube were 4.45mg/mL ±0.35 and 4,52mg/mL ±0.29 for right and left eyes respectively. The average protein concentration and standard deviation from tears collected with Schirmer Tear Test (STT) strip were and 54.5mg/mL ±0.63 and 54.15mg/mL ±0.65 to right and left eyes respectively. Statistically significant differences (p<0.001) were found between the methods. In the conditions in which this study was conducted, the average protein concentration obtained with the Bradford test from tear samples obtained by Schirmer Tear Test (STT) strip showed values higher than those obtained with microcapillary tube. It is important that concentration of tear protein pattern values should be analyzed according the method used to collect tear samples.

Compararam-se dois métodos de coleta de lágrima para quantificação proteica, utilizando-se 58 olhos de 29 cães hígidos. As amostras foram coletadas utilizando-se fitas de teste da lágrima de Schirmer (Schirmer tear test - STT) e tubos microcapilares. Após obtidas, as amostras foram congeladas a -80º C e posteriormente analisadas pelo método de Bradford. Os resultados foram analisados pelo teste T de Student. A média da concentração proteica e desvio padrão das amostras obtidas com microcapilar foi de 4,45mg/mL ±0,35 e 4,52mg/mL ±0,29 para olhos direito e esquerdo, respectivamente. Para as amostras obtidas com STT os resultados foram 4,45mg/mL ±0,35 and 4,52mg/mL ±0,29 para olhos direito e esquerdo, respectivamente. Diferenças estatisticamente significativas (p<0,001) foram encontradas entre os dois métodos. Nas condições em que o trabalho foi conduzido, a média da concentração proteica pelo método de Bradford das amostras obtidas através das tiras do STT foi superior à obtida com o tubo microcapilar. Os valores padrão da concentração protéica da lágrima obtida pelo método de Bradford devem ser analisados considerando-se o método de coleta da lágrima, uma vez que este interfere significativamente nos resultados obtidos.