A Novel Assay For Detecting Serum Proteins In Tear Fluid: Diagnostic Implications In Dry Eye Associated With Systemic Immunological Diseases.

Abstract

Purpose : It is known that serum proteins leak from conjunctival vessels, admix with the tear fluid, and influence the tear film biophysical properties like stability and surface tension. We have developed a florescence probe (FP) assay to determine the presence of serum proteins in 1μL of tear fluid. We used the FP assay in patients with immunological or non-immunological dry eye disease (DED) to determine whether it can differentiate between these DED subtypes and whether higher assay readings are associated with the presence of inflammatory cells in the tear fluid.

Methods : Tear fluid was collected from patients with two distinct DED subtypes: (i) Immunological DED that includes Sjogren’s syndrome (n=12) and Graft-versus-host-disease (GVHD; n= 16); and (ii) Non-immunological DED that includes Meibomian gland dysfunction (MGD; n=22) and non-autoimmune DED (n=15). Healthy volunteers (n=21) served as controls. Tear liquid (1μL) and FP (1 μL) were mixed and florescence intensity was measured at excitation wavelength of 430 and emission wavelength of 490 using Take3 platform and Cytation 5 Cell Imaging Multi-Mode Reader (BioTek, Winooski, VT). The number of cells in tear fluid (3μL) was determined using AO/PI staining with automated Cellometer K2 (Nexcelom,MA). Tear fluid cells were immunostained with neutrophil elastase and keratin 14 to identify inflammatory and ocular surface cells, respectively.

Results : In healthy volunteers, florescence intensity was measured 6575.7 +/- 1982.3. Florescence intensity was significantly higher in immunological DED (53603.2 +/- 13716.2 in Sjogren’s syndrome and 37447.5 +/- 15653.6 in GVHD; p<0.05), but not in non-immunological DED (8692.5 +/- 7729.3 in MGD; p>0.05).The presence of tear fluid cells was also significantly greater in immunological DED. These cells were predominantly neutrophils in immunological DED and exfoliated corneal cells in non-immunological DED.

Conclusions : Patients with immunological DED have significantly greater serum proteins and neutrophils in their tear fluid. The presence of these blood components in the tear fluid points to increased vascular leakage in immunological DED. The FP tear fluid assay may be helpful in early detection of vascular leakage and may point to the presence of a systemic immunological condition associated with DED.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.