Adaptation of impression cytology to enable conjunctival surface cell transcriptome analysis.

PURPOSE: This study investigates the extent of the human transcriptome that can be quantified from conjunctival impression cytology extracts. The aim is to determine if sufficient RNA can be isolated from a patient's conjunctival surface to identify differences in gene expression between dry eye and normal patients of (a) an array of 96 inflammatory biomarkers and associated receptors, and (b) if this comparison can be expanded to the entire transcriptome. MATERIALS AND METHODS: CIC was used to collect conjunctival surface cells from 53 qualifying normal and dry eye patients. Based on prior optimization of all assay steps, RNA was isolated from the samples using a Qiagen RNeasy Plus Mini Kit and qRT-PCR was used to determine gene expression of 96 genes using TaqMan Low Density Array cards. Samples from six normal and six dry eye patients were then assayed on an Illumina Human HT-12 BeadChip. RESULTS: Optimization steps yielded an RNA processing procedure that improved yield from an initial 12 genes through 96, then to the entire human transcriptome. For the HT-12 BeadChip, more than 30 genes differed by a factor of >1.5 between the dry eye and normal groups and seven genes were down-regulated by a factor of >2.0 in the dry eye group: HLA-DRB5, PSCA, FOS, lysozyme, TSC22D1, CAPN13 and CXCL6. CONCLUSIONS: Conjunctival impression cytology can be used to collect sufficient RNA from conjunctival surface cells that, when processed optimally, allows successful transcriptome-wide expression analysis. While the current transcriptome analysis used a limited patient group, larger studies of patients with various types and severities of dry eye should reveal significant gene expression trends that can then be targeted to improve dry eye treatment options.

Comparison of low-abundance biomarker levels in capillary-collected nonstimulated tears and washout tears of aqueous-deficient and normal patients.

PURPOSE: Low tear volume limits the use of nonstimulated (NS) microcapillary tear collection in aqueous-deficient (AD) patients. Adding a small amount of "washout" fluid to the eye prior to tear collection is a potentially viable alternative method for abundant proteins, but is relatively untested for low-abundance biomarkers. This study determined the feasibility of the washout (WO) method as an NS alternative for low-abundance biomarkers. NS and WO biomarker profiles were compared between AD patients and non-AD controls to determine if the two methods identify the same intergroup differences. METHODS: Matching NS and WO tears were collected from 48 patients by micropipette, the WO sample after instillation of 10 µL saline. Tear cytokine levels were measured by 27-Plex Bio-Rad assay. Bland-Altman analyses for each biomarker determined the agreement between tear sample types. Patients were grouped as AD or non-AD based on Schirmer score to determine if NS profile between-group differences were preserved in WO tears. RESULTS: Bland-Altman plots showed good biomarker level agreement between NS and WO tears for most cytokines. Five biomarkers, among those most often cited as differing in AD dry eye, differed significantly between non-AD and AD groups in both tear types. Additional biomarker differences were seen in NS tears only. CONCLUSIONS: The WO tear collection method is a viable alternative to NS tears for many low-abundance biomarkers and is able to replicate major NS tear differences between dry eye groups. More subtle intergroup differences are lost in WO samples because of reduced statistical power.

Aniseikonia testing in an adult population using a new computerized test, "the Aniseikonia Inspector".

PURPOSE: To determine the measurement characteristics of a new computerized test, the Aniseikonia Inspector Version 1, on a sample of clinic patients. METHODS: Aniseikonia was measured in the vertical, horizontal, and oblique meridians on 320 patients (mean 55 years old, range 17-89 years) prior to their optometric exam using the psychometric methods programmed into the Aniseikonia Inspector Version 1. Statistical analyses were performed to determine the distribution of aniseikonia in the sample of patients and the relationships between the amount of aniseikonia and patients' habitual refractive correction, visual acuity, stereopsis and binocular alignment status. The characteristics of the individual measurements were also examined. RESULTS: The means and standard deviations of the measured aniseikonia in the vertical, horizontal, and oblique meridians were -0.5% (2.5%), -0.1% (3.3%) and 0.3% (2.8%) respectively. The means in the vertical and oblique meridians were significantly different from 0.0 (p=0.0001, p=0.0314) while that in the horizontal was not (p=0.61). The distributions of aniseikonia showed that 65.6%, 57.5% and 64.3% had within +/-1.0% aniseikonia in the vertical, horizontal and oblique meridians, respectively. Correspondingly, 16.9%, 25.6% and 25.8% had aniseikonia of +/-3.0% or greater. The discrepancy between these percentages and those expected in a normal distribution indicate that the distributions were significantly more peaked than a normal distribution. This departure from normal is due to a few extreme values in the tails. The magnitude of aniseikonia had no statistically significant relationship with the patients' habitual refractive correction, visual acuity or stereopsis. The effect of phoria on the amount of aniseikonia was significant, more so for measurements in the horizontal meridian. The individual measurements, which are the average of two trials using the method of adjustment, showed no significant bias, no relationship between the means and differences in the two readings, but large differences between the two readings. Measurements in the vertical direction seem to be more stable than those in the other two meridians. CONCLUSION: As measured with the Aniseikonia Inspector 1.0, the majority of the patients sampled in this study exhibited 1.0% or less aniseikonia and were therefore not likely to have symptoms related to aniseikonia. At least 17% of patients had 3.0% or greater aniseikonia measured in the vertical meridian. The Aniseikonia Inspector warrants further evaluation in a clinical setting because of the large limits of agreement between the two settings that are average to determine the magnitude of the aniseikonia. These limits differ considerably from those established by the designers and, therefore, raise questions regarding the actual resolution of the instrument as compared to the nominal resolution.