Dr.Pooja Khamar

Dr. ROHIT SHETTY

Abstract

Purpose: To develop a rapid, multiplexed diagnostic kit, to evaluate the Molecular levels in tears of patients undergoing Refractive Surgery, Suspect Keratoconus(KC) eyes and Dry eye Disease treatment(DED).

Methods: 28 controls, 20 KC/post-refractive surgery ectasia, 30 DED, were measured. Tear samples using schirmer’s strips were added to the test cartridge and inserted into a laser based detection platform to determine the concentration of the analyte on the basis of a standard curve.

Results: IL6, IL8, MMP9 were higher(p<0.05) in DED and KC. LOX levels were lower (p<0.05) in cases of post-surgery ectasia(pre-op topography and biomechanic:normal). The multiplexed kit successfully detected all analytes demonstrating the highest fold change and significance.

Conclusion: Tear point-of-care diagnostic kit can thus revolutionize screening protocols in refractive surgery and other diseases. This can enable better patient care by predicting outcomes and customise treatments by clinician.

Full Text

Introduction:

Higher levels of inflammatory factors(IL-6, IL-8, MMP-9), result in dry eye disease (DED) and may have implications for recovery of patients undergoing refractive surgery. We therefore, tested the efficiency of simple, rapid multi-analyte tear based system for measuring multiple inflammatory factors for clinical applications.

Methods:

Tear samples were collected from 50 healthy controls, 50 patients with ocular surface conditions and 10 patients with intra-ocular conditions (glaucoma, diabetic retinopathy) with prior informed consent and institutional ethics approval. Using a variant of the multiplex ELISA assay system, we obtained specific cartridges for IL-6, IL-8 and MMP-9 measurement in a single test. The Schirmer’s strips were collected in 1.5ml tubes containing 300µl extraction buffer followed by agitation for 15-30 min. 50µl of the resulting extract was added to each sample well of the cartridge. 1 ml of the specific wash buffer was added to the designated buffer well. The cartridge was loaded into the analyser instrument which took 90 minutes to deliver the measured values on the basis of established internal references for each analyte.

Results:

All three analytes (IL-6, IL-8 and MMP-9) were significantly(P<0.05) higher in ocular surface conditions. The instrument generated RFU (relative fluorescence units) values from each sample were normalised to the wetting length. IL6 and MMP9 were log-fold higher in dry eye disease (DED) and Steven-Johnson syndrome (SJS) samples (P<0.05) while ocular surface chemical injury samples only had high levels of IL8 (P<0.05). IL8 levels were slightly elevated in case of the non-ocular surface conditions glaucoma and diabetic retinopathy, which has several fold lesser than that observed for DED and SJS. The data demonstrated detection of all three analytes in the 50µl sample that allowed for sufficient sample remaining if reruns are required.

Conclusion: The data demonstrates that IL6, MMP9 and IL8 levels were detectable across the entire set of patient samples and show clear disease based separation of values. The efficiency and short test duration can significantly assist screening protocols in dry eye and refractive surgery to enable better patient stratification.