Vatinee Y. Bunya

Isolation of cell surface-specific human monoclonal antibodies using phage display and magnetically-activated cell sorting: applications in immunohematology.

A method is described for the isolation of filamentous phage-displayed human monoclonal antibodies directed at unpurifiable cell surface-expressed molecules. To optimize the capture of antigen-specific phage and minimize the binding of irrelevant phage antibodies, a simultaneous positive and negative selection strategy is employed. Cells bearing the antigen of interest are pre-coated with magnetic beads and diluted into an excess of unmodified antigen-negative cells. Following incubation of the cell admixture with a Fab/phage library, the antigen-positive cell population is retrieved using magnetically-activated cell sorting and antigen-specific Fab/phage are eluted and propagated in bacterial culture. Utilizing this protocol with magnetically-labeled Rh(D)-positive and excess unlabeled Rh(D)-negative human red blood cells and a Fab/phage library constructed from human peripheral blood lymphocytes, dozens of unique clinically-useful gamma 1 kappa and gamma 1 lambda anti-Rh(D) antibodies were isolated from a single alloimmunized individual. This cell-surface selection method is readily adaptable for use in other systems, such as for the identification of putative tumor-specific antigens and provides a rapid (< 1 month), high-yield approach for isolating self-replicative antibody reagents directed at novel or conformationally-dependent cell-surface epitopes.

TFOS DEWS II Epidemiology Report

The subcommittee reviewed the prevalence, incidence, risk factors, natural history, morbidity and questionnaires reported in epidemiological studies of dry eye disease (DED). A meta-analysis of published prevalence data estimated the impact of age and sex. Global mapping of prevalence was undertaken. The prevalence of DED ranged from 5 to 50%. The prevalence of signs was higher and more variable than symptoms. There were limited prevalence studies in youth and in populations south of the equator. The meta-analysis confirmed that prevalence increases with age, however signs showed a greater increase per decade than symptoms. Women have a higher prevalence of DED than men, although differences become significant only with age. Risk factors were categorized as modifiable/non-modifiable, and as consistent, probable or inconclusive. Asian ethnicity was a mostly consistent risk factor. The economic burden and impact of DED on vision, quality of life, work productivity, psychological and physical impact of pain, are considerable, particularly costs due to reduced work productivity. Questionnaires used to evaluate DED vary in their utility. Future research should establish the prevalence of disease of varying severity, the incidence in different populations and potential risk factors such as youth and digital device usage. Geospatial mapping might elucidate the impact of climate, environment and socioeconomic factors. Given the limited study of the natural history of treated and untreated DED, this remains an important area for future research.

Variability of Tear Osmolarity in Patients With Dry Eye

IMPORTANCE Knowledge about the variability of measurements using the TearLab Osmolarity System is necessary when evaluating the clinical utility of readings. OBJECTIVE To examine the variability of tear osmolarity measured by the TearLab Osmolarity System in patients with Sjögren syndrome (SS), patients with blepharitis, and control participants. DESIGN, SETTING, AND PARTICIPANTS Cross-sectional study at a tertiary care academic center from June 13, 2012, to March 21, 2013. Participants included 74 eyes of 37 patients from a volunteer sample (18 patients with SS, 11 patients with blepharitis, and 8 control participants) who were evaluated using the TearLab Osmolarity System, with 3 consecutive osmolarity measurements taken at 1-minute intervals in a session; 15 of these patients had the same measurements taken by the same examiner in 2 additional sessions on the same day (9 AM-10 AM, 12 PM-1 PM, or 3 PM-4 PM). Most patients with SS and patients with blepharitis were taking systemic or topical dry eye medications at the time of enrollment. MAIN OUTCOMES AND MEASURES Mean osmolarity and its variability calculated from a linear mixed model for each disease group that accounts for the variations attributable to different patients, eyes, and sessions and measurement error specific to each disease group. RESULTS Mean tear osmolarity was 307 mOsm/L, 304 mOsm/L, and 301 mOsm/L in the SS, blepharitis, and control groups, respectively (P = .46). The error associated with repeated measurements within a session in the patients without dry eye (10.5 mOsm/L [95% CI, 9.0-12.4]) was significantly lower than in the patients with blepharitis (14.6 mOsm/L [95% CI, 12.5-17.5]; P = .006) and patients with SS (15.8 mOsm/L [95% CI, 14.2-17.8]; P < .001) but a difference in the error of repeated measurements between patients with blepharitis and patients with SS was not identified (P = .46). CONCLUSIONS AND RELEVANCE There was increased variability attributable to error in repeated measurements in patients with SS and patients with blepharitis compared with control participants. The high variability of TearLab osmolarity readings in all groups makes the clinical interpretation of measurements unclear.

Corneal edema and haze after selective laser trabeculoplasty.

PurposeTo report 2 cases of corneal edema, haze, and thinning in patients after undergoing selective laser trabeculopasty. MethodsSelective laser trabeculoplasty was performed for the treatment of primary open-angle glaucoma on 2 patients who subsequently developed corneal stromal haze within 24 to 48 hours of the procedure. ResultsThe patients were treated with topical steroids for several weeks. Although their corneal edema resolved, both patients were left with residual corneal scarring and thinning. One patient had a significant hyperopic shift. ConclusionsCorneal edema, haze, and thinning after selective laser trabeculoplasty is an extremely rare event, with only 2 other cases reported in the literature. Although certain causes are postulated to play a role in this complication, it is not yet understood what may predispose a patient to corneal changes as a result of this laser procedure.

Tear Osmolarity and Dry Eye Symptoms in Women Using Oral Contraception and Contact Lenses

Purpose: The aim of this study was to examine the relationship among oral contraceptive pill (OCP) use, contact lens wear, and dry eye signs and symptoms in healthy young women. Methods: Fifty-two women using OCPs and 45 women not using any form of hormonal contraception were enrolled. Medical, menstrual, and contact lens histories were obtained and dry eye symptoms assessed using the Ocular Surface Disease Index (OSDI) and Symptom Assessment iN Dry Eye (SANDE) questionnaires. Tear osmolarity testing was performed using the TearLab Osmolarity System. Results: The mean age of all subjects was 26.0 ± 3.7 years. There were no significant differences in any of the measurements between the follicular and luteal phases. Although SANDE scores were significantly higher in subjects with OCP and recent contact lens use (P < 0.01), there were no significant differences in OSDI and tear osmolarity among the same subject groups. Subjects who reported both OCP and recent contact lens use had significantly higher OSDI and SANDE scores (P = 0.015 and P < 0.001, respectively). Conclusions: There were no differences between the phases of the menstrual cycle. Tear osmolarity was not affected by OCP or contact lens use in young women. However, the combination of OCP use and contact lens wear may increase the severity of dry eye symptoms.

The SSB-positive/SSA-negative antibody profile is not associated with key phenotypic features of Sjögren's syndrome

Objective To determine whether the Sjögrens syndrome B (SSB)-positive/Sjögrens syndrome A (SSA)-negative antibody profile is associated with key phenotypic features of SS. Methods Among registrants in the Sjögrens International Collaborative Clinical Alliance (SICCA) with possible or established SS, we compared anti-SSA/anti-SSB reactivity profiles against concurrent phenotypic features. We fitted logistic regression models to explore the association between anti-SSA/anti-SSB reactivity profile and each key SS phenotypic feature, controlling for potential confounders. Results Among 3297 participants, 2061 (63%) had negative anti-SSA/anti-SSB, 1162 (35%) had anti-SSA with or without anti-SSB, and 74 (2%) anti-SSB alone. Key SS phenotypic features were more prevalent and had measures indicative of greater disease activity in those participants with anti-SSA, either alone or with anti-SSB, than in those with anti-SSB alone or negative SSA/SSB serology. These between-group differences were highly significant and not explained by confounding by age, race/ethnicity or gender. Participants with anti-SSB alone were comparable to those with negative SSA/SSB serology in their association with these key phenotypic features. Among SICCA participants classified with SS on the basis of the American-European Consensus Group or American College of Rheumatology criteria, only 2% required the anti-SSB-alone test result to meet these criteria. Conclusions The presence of anti-SSB, without anti-SSA antibodies, had no significant association with SS phenotypic features, relative to seronegative participants. The solitary presence of anti-SSB antibodies does not provide any more support than negative serology for the diagnosis of SS. This serological profile should thus be interpreted cautiously in clinical practice and potentially eliminated from future classification criteria.

Tear osmolarity in Sjögren syndrome.

Purpose: The Schirmer test is one of the 2 ocular surface tests included in the current classification criteria for Sjögren syndrome (SS). Tear osmolarity may also be a useful test for the diagnosis of dry eye disease. The purpose of this study was to examine the relationship between tear osmolarity, the Schirmer test I, and dry eye symptoms in SS. Methods: Patients with a diagnosis of SS were assessed for tear osmolarity with the TearLab Osmolarity System, tear production with Schirmer testing, symptoms with the Ocular Surface Disease Index (OSDI), and discomfort associated with each test. Results: Forty-nine patients with a mean age of 53.7 years and a female (92%) predominance were enrolled. The majority of patients (86%) were receiving systemic therapy for severe SS. Higher tear osmolarity was moderately associated with lower scores on the Schirmer test I (&rgr; = −0.39, P < 0.01) and OSDI (&rgr; = −0.45, P < 0.01). Schirmer test I results and lower OSDI scores were not correlated significantly (&rgr; = 0.20, P = 0.17). Tear osmolarity testing was significantly less painful than Schirmer testing (P < 0.01). Conclusions: Signs and symptoms of dry eye in SS patients were not strongly correlated. An unexpected finding was that higher tear osmolarity was associated with lower symptom severity. Tear osmolarity testing in the clinical setting was feasible and was associated with significantly less discomfort than Schirmer testing in patients with severe SS.

Tear osmolarity and dry eye symptoms in diabetics

Purpose To assess the relationship between tear osmolarity and dry eye symptoms in patients with diabetes. Patients and methods Fifty patients with diabetes were enrolled. Demographic information and past medical history were recorded. Symptoms were assessed using the ocular surface disease index (OSDI). Tear osmolarity of each eye was measured with the TearLab® Osmolarity System. Results The majority of the subjects were female (76%), African American (56%), and/or had a diagnosis of type 2 diabetes (82%). The mean ± standard deviation (SD) for age was 54.6±13.4, and maximum tear osmolarity was 304.6±12.7 mOsm/L. Men had higher osmolarity than women (mean ± standard error (SE) 311.8±4.0 mOsm/L versus 302.3±1.9 mOsm/L, P=0.02). Age, race, use of artificial tears, years of diabetes, and hemoglobin A1c did not have a statistically significant association with tear osmolarity. Longer duration of diabetes was associated with lower (less severe) OSDI scores (r=−0.35, P=0.01). Higher tear osmolarity was associated with lower (less severe) OSDI scores (r=−0.29, P=0.04). Conclusion Approximately half of the diabetic subjects in our study had elevated tear osmolarity, and half of our population also reported symptoms consistent with dry eye disease. However, the two were slightly inversely related in that those with higher osmolarity reported fewer symptoms. Subjects with a longer duration of diabetes also reported fewer dry eye symptoms. Therefore, health care providers should be aware that patients who are most likely to have ocular surface disease, including those with long-standing diabetes, may not experience symptoms and seek care in a timely manner.

Inter-grader Agreement of the Ocular Staining Score in the Sjögren's International Clinical Collaborative Alliance (SICCA) Registry.

PURPOSE To determine the intra-observer and inter-observer reliability of a novel ocular staining score among trained ophthalmologists. DESIGN Reliability analysis within a prospective, observational, multicenter cohort study. METHODS Those enrolled in the National Institutes of Health-funded Sjögrens International Collaborative Clinical Alliance (SICCA) who presented for follow-up at the University of California San Francisco, Aravind Eye Hospital, Johns Hopkins University, and the University of Pennsylvania were included. Study participants were graded using the ocular staining score by at least 2 masked SICCA-trained ophthalmologists. The primary outcome for this study was the intraclass correlation coefficient (ICC) for the total ocular staining score. ICCs were also calculated for tear break-up time (TBUT) and conjunctival and corneal staining. RESULTS Total ocular staining score had an ICC of 0.91 for the right eye (95% confidence interval [CI] 0.85-0.96) and 0.90 for the left eye (95% CI 0.83-0.97). Corneal staining (right eye 0.86, 95% CI 0.76-0.93, left eye 0.90, 95% CI 0.81-0.95) and conjunctival staining (right eye 0.87, 95% CI 0.80-0.93, left eye 0.85, 95% CI 0.75-0.93) demonstrated excellent agreement. The ICC for TBUT was slightly lower (right eye 0.77, 95% CI 0.64-0.89; left eye 0.81, 95% CI 0.68-0.90). CONCLUSIONS Previous studies have shown that the ocular staining score is correlated with other diagnostic components of Sjögren syndrome. In this study, we demonstrate high reliability in grading among trained ophthalmologists, completing the validation of this test.

Elevated intraocular pressure-induced interlamellar stromal keratitis occurring 9 years after laser in situ keratomileusis.

Elevated intraocular pressure-induced interlamellar stromal keratitis (PISK) is an entity of interface haze usually occurring weeks to months after laser in situ keratomileusis (LASIK) that is associated with elevated intraocular pressures and worsening with steroid treatment. There is evidence that this interface haze is the result of abnormal fluid dynamics that occur in the cornea after LASIK. We present a case of pressure-induced interlamellar stromal keratitis occurring 9 years after LASIK in the setting of anterior uveitis. This case emphasizes the importance of considering such diagnoses as pressure-induced interlamellar stromal keratitis in the differential diagnosis when presented with a patient with corneal haze and a history of LASIK.