title: Limitations of an ocular surface inflammatory biomarker in impression cytology specimens.
abstract: BACKGROUND
  A number of ocular conditions, such as dry eye, are associated with inflammation on the surface of the eye leading to irritation and ocular pain. Many drugs such as chemotherapeutics, beta blockers, angiotensin-converting enzymes and so forth also cause dry eye but currently there are no validated ocular surface biomarkers available.
  
  
  OBJECTIVE
  We evaluated sample stability, assay sensitivity, reproducibility and overall performance of impression cytology (IC) utilizing the cellular surface biomarker human leukocyte antigen DR-1 (HLA-DR) as an ocular surface inflammatory biomarker by flow cytometry in a fit-for-purpose validation study. Additionally, subjects classified as normal or having various degrees of dry eye were evaluated to determine if HLA-DR could demonstrate a clear separation between normal and dry eye samples.
  
  
  RESULTS
  The assay demonstrated high dynamic range detecting a broad range of fluorescent intensities in healthy donors. Additionally, inter, intra and stability assay results demonstrated strong concordance and low variability. Overall CV% for both assays were less than 25% for all measured parameters. However, high variability was observed for donor samples assayed beyond day 10 post IC sample collection (4.2-110.8 CV%).
  
  
  CONCLUSIONS
  HLA-DR expression demonstrated a progressive increase in patients with mild to severe levels of dry eye disease providing sufficient evidence it is sensitive enough to monitor inflammatory effects of dry eye when coupled with additional biomarkers and/or methodologies such as cytokine analysis or ICAM-1. This biomarker can be used to monitor ocular surface disorders in patients and to evaluate potential treatment options during drug development. Although our results demonstrate this methodology is reproducible for routine evaluation, limitations around sample integrity exist.
  
  
  CONCLUSIONS
  The ocular cell surface inflammatory biomarker, HLA-DR coupled with impression cytology is a simple non-invasive robust, specific and reproducible assay that can be utilized to measure inflammatory infiltrates on the surface of the eye in IC samples less than 10-days old.
authors.affiliation: Pfizer Global Research and Development, Drug Safety Research and Development, Biomarkers, San Diego, CA 91915, USA.
authors.forename: Rolla
authors.initials: R
authors.lastname: Yafawi
authors.forename: Mira
authors.initials: M
authors.lastname: Ko
authors.forename: Frederick P
authors.initials: FP
authors.lastname: Sace
authors.forename: Annette
authors.initials: A
authors.lastname: John-Baptiste
mesh_terms: D000328:Adult; D015415:Biomarkers / Q000378:metabolism; D003228:Conjunctiva / Q000166:cytology; D015352:Dry Eye Syndromes / Q000175:diagnosis* / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods; D006684:HLA-DR Antigens / Q000378:metabolism*; D006801:Humans; D007249:Inflammation / Q000175:diagnosis / Q000378:metabolism
chemical_list: D015415:Biomarkers; D006684:HLA-DR Antigens