Abstract
Purpose:
To establish a standardized methodology for determining the relative amount of HLA-DR on ocular surface cells for use as a minimally invasive objective metric for large scale multicenter clinical trials and translational research studies of ocular surface disease.
Methods:
To establish a Standard Operating Procedure (SOP) for utilizing HLA-DR expression as a biomarker of inflammation of the ocular surface the following was done: duplicate cell samples from impression cytology (IC) samples of both normal and dry eye (DE) individuals were collected and split to assess repeatability. To determine storage capability, one duplicate was stained immediately and the other after 30 days. To demonstrate the feasibility of the use of the SOP for a multicenter clinical trial, clinicians from out-of-state were trained to collect IC samples which were shipped to New York for processing and analysis by a masked independent observer. IC and HLA-DR quantification were incorporated into a masked randomized clinical trial of DE.
Results:
The validity/viability of the SOPs was established: 1) sufficient numbers of cells can be collected via IC (average: 6,677 ± 6,302 cells/filter (normal: 8,511 ± 7,743, n = 114, range: 1,389 − 101,031; DED: 5,414 ± 5,128, n = 252, range: 1,032 − 58,892). 2) relative biomarker expression quantified in samples is precise/repeatable (mean difference: −0.12%, p = 0.23; 95% limits of agreement for splits: −0.82%, +0.58%); 3) personnel at distant sites can be taught to collect, store and ship samples; 4) samples can be stored until processing can be performed without affecting results [mean difference for HLA-DR%: 0.31% (ie, Day 30 values were on average higher by 0.31%; p = 0.11); 95% limits of agreement were (−0.91%, +1.53%)]; 5) a large number of masked samples can be reliably tracked.
Conclusions:
We demonstrated the repeatability and effects of storage, ability to train/gather samples from distant sites and the feasibility of use in a randomized clinical trial. HLA-DR expression as determined from IC samples can serve as a minimally invasive objective metric of inflammation for efficacy and mechanism of action in randomized clinical trials of ocular surface disease.
Keywords: 557 inflammation •
529 flow cytometry •
486 cornea: tears/tear film/dry eye