Internal Standard Dilution Indicator Protocol (ISDIP): A Newer Method for the Quantitative Assessment in Tear Lipidomic Studies

Abstract

Purpose: Sampling tear in patients suffering from tear deficiency disorders for the quantitative assessment of proteomic or lipidomic studies is challenging task due to the sampling errors due to low volume of aqueous phase. This study developed and validated in healthy human volunteers, a new tear collection method ISDIP for its utilisation in patients with dry eye.

Methods: The study protocol was approved by the Standing Institutional Human Ethics Committee, AIIMS and was followed Helsinki. Informed consent was obtained from all volunteers. Both internal standard (IS) and decoy marker for validation were selected from the approved chemicals for ocular use. Tear samples were collected from healthy volunteers using 3 different protocols; Standard capillary, Schirmer’s strip and ISDIP methods. All the volunteers were subjected for TBUT and Schirmer’s test 1 score. ISDIP was used 50 µl of IS (0.005% w/v with 1.4% PVA) instilled topically and instructed to volunteers to close their eye slowly and rotate them to give enough time for tear extraction. Tear samples were collected after 15sec by sterilized fire polished glass microcapillary from outer fornix. 20µl decoy marker (ofloxacin) was instilled topically 5min prior to tear collection. Decoy marker and IS levels were analyzed by LC-MS/MS in tears. The samples collected by ISDIP from healthy eyes were subjected for the selected polar and non-polar lipid quantification.

Results: Tear was collected from healthy volunteers (13 male and 8 female with mean age 29.15±5.49 and 25.87±3.56yrs respectively). Tear decoy marker level were found to be 10.6 ± 4.4 μg/ml by ISDIP in healthy volunteers and were found to be higher than other two methods employed. The concentration of decoy marker was found to be significant Pearson correlation (p<0.05) with TBUT score of volunteers in ISDIP and there was no such correlation observed with the samples collected from other two methods.

Conclusions: The newly developed method (ISDIP) for tear collection is capable of removing the errors like reflux tearing, lack of adequate sampling in dry eye, tear evaporation while collection etc.. By using this method we can able to collect adequate amount of tear constituents from patients suffering with ocular surface disorder like dry eye. Therefore, ISDIP might be helpful in tear omics quantitative studies in severe dry eye condition.

Keywords: 583 lipids • 479 cornea: clinical science • 683 refractive surgery: LASIK