Tear cytokine profile as a noninvasive biomarker of inflammation for ocular surface diseases: standard operating procedures

Abstract

Purpose: To provide standard operating procedures (SOPs) for measuring tear inflammatory cytokine concentrations and to validate the resulting profile as a minimally invasive objective metric and biomarker of ocular surface inflammation for use in multicenter clinical trials on dry eye disease (DED).

Methods: Standard operating procedures were established and then validated with cytokine standards, quality controls, and masked tear samples collected from local and distant clinical sites. The concentrations of the inflammatory cytokines in tears were quantified using a high-sensitivity human cytokine multiplex kit.

Results: A panel of inflammatory cytokines was initially investigated, from which four key inflammatory cytokines (IL-1β, IL-6, INF-γ, and TNF-α) were chosen. Results with cytokine standards statistically satisfied the manufacturer's quality control criteria. Results with pooled tear samples were highly reproducible and reliable with tear volumes ranging from 4 to 10 μL. Incorporation of the SOPs into clinical trials was subsequently validated. Tear samples were collected at a distant clinical site, stored, and shipped to our Biomarker Laboratory, where a masked analysis of the four tear cytokines was successfully performed. Tear samples were also collected from a feasibility study on DED. Inflammatory cytokine concentrations were decreased in tears of subjects who received anti-inflammatory treatment.

Conclusions: Standard operating procedures for human tear cytokine assessment suitable for multicenter clinical trials were established. Tear cytokine profiling using these SOPs may provide objective metrics useful for diagnosing, classifying, and analyzing treatment efficacy in inflammatory conditions of the ocular surface, which may further elucidate the mechanisms involved in the pathogenesis of ocular surface disease.

Keywords: dry eye disease; milliplex assay; standard operating procedures; tear cytokine profile.

Figure 1

Standard curves of four cytokines of one representative experiment. The standard curves of IL-1β, IL-6, IFN-γ, and TNF-α were plotted by xPONENT 3.1 software (Luminex Corporation, Austin, TX) using the MFI against the concentrations of indicated analyte. The coefficient of variance, the R² for linearity regression, and minimal–maximal detection ranges given by xPONENT software are indicated. The ranges from 0.64 to 400 pg/mL points are boxed, and the points at 0.64 pg/mL are circled as reference points. The slope between MFI baseline and the log-linear phase line is also outlined.

Figure 2

Tear dilution effect and minimal tear volume (MTV) determination. Two pooled tear samples (A and B) were each analyzed with 2, 4, 6, 8, or 10 μL tears brought up to 50 μL by the assay buffer; each dilution was measured in seven (A) or eight (B) repeats. The mean concentrations of four selected cytokines were plotted against the tear volumes used in assay. Error bars represent the standard deviations of the mean. Multiple Student's t-tests were performed to compare the statistical differences among dilutions; the results with P values < 0.05 are marked with asterisks.

Figure 3

Tear cytokine levels in DED patients before and after 3 months of anti-inflammatory drug treatment. Tears pooled from the left (OS) and right (OD) eye of each DED subject in an IRB- and FDA-approved double-blind RCT study of systemic ω3 treatments were analyzed per SOPs. Patients received either ω3 or placebo (plcb) at randomized base. The concentrations of four selected cytokines were subsequently evaluated at baseline (pre-plcb or pre-ω3) and after 3 months of treatment (post-plcb or post-ω3). Changes of cytokine concentrations were calculated as shown on top of each figure. Trend of changes is highlighted by red arrows (with changes) and green arrows (no changes). The numbers (n) of patients per treatment groups are indicated.

Figure 4

Scheme of SOPs. Critical steps to ensure reliable results are as follows: (1) tear collection by trained personnel with careful attention to masked labeling; (2) proper shipping of samples; (3) proper storage and tracking of samples with backup; (4) analysis with controls utilizing the MILLIPLEX MAP High Sensitivity Human Cytokine Magnetic Bead Panel; (5) accurate recording of masked results; (6) secure data collecting and transferring to the coordinating center for unmasking, analysis, and correlation with other clinical and laboratory findings. Serious consideration should also be given to the following: (1) including all commercial standards and reference controls (QC1 and 2) as well as additional tear aliquots (QC 3) for use over the length of the clinical trial with reasonable replicates; (2) utilizing assay kits from the same product lot of the same manufacturer—if a trial continues over an extended period (years), new lots of all kits must be regularly compared and standardized to those previously used; (3) calibrating pipettes periodically and standardizing the tips, tubes, and liquid transfer methods; (4) keeping sample and data tracking/logging records on a regular basis at every step, ideally with several cross-checking and secured backup systems running simultaneously; (5) creating a monthly report to the coordinating center and all of the participating clinical sites to provide quality control metrics ensuring compliance with SOPs; (6) deidentifying data only by the authorized personnel from the coordinating center.