title: Tear cytokine profile as a noninvasive biomarker of inflammation for ocular surface diseases: standard operating procedures.
abstract: OBJECTIVE
  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.
authors.affiliation: Department of Ophthalmology, Icahn School of Medicine at Mount Sinai, New York, New York.
authors.forename: Yi
authors.initials: Y
authors.lastname: Wei
authors.forename: Neha
authors.initials: N
authors.lastname: Gadaria-Rathod
authors.forename: Seth
authors.initials: S
authors.lastname: Epstein
authors.forename: Penny
authors.initials: P
authors.lastname: Asbell
mesh_terms: D015415:Biomarkers / Q000378:metabolism*; D016207:Cytokines / Q000378:metabolism*; D004311:Double-Blind Method; D015352:Dry Eye Syndromes / Q000378:metabolism*; D006801:Humans; D007249:Inflammation / Q000378:metabolism*; D007371:Interferon-gamma / Q000378:metabolism; D053583:Interleukin-1beta / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D008875:Middle Aged; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D011795:Surveys and Questionnaires; D013666:Tears / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D015415:Biomarkers; D016207:Cytokines; C508600:IL6 protein, human; D053583:Interleukin-1beta; D015850:Interleukin-6; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma
keywords: dry eye disease; milliplex assay; standard operating procedures; tear cytokine profile