Correlation between mRNA and protein expression profiles of HLA-DR in Conjunctival Impression Cytology using a new device for collecting epithelial cells

Abstract

Purpose: Human Leucocyte Antigen (HLA-DR) expression in conjunctival impression cytology (CIC) is recognized as a reliable biomarker to follow various inflammatory ocular surface (OS) disorders. Flow Cytometry (FC) is routinely used to investigate the inflammatory level of OS through a quantification of HLA-DR antigen expression. Our aim was, using the new cell collection device EyePRIM (EP), to compare two different approaches for HLA-DR evaluation: FC for the surface protein assessment and Real time Polymerase Chain Reaction (PCR) for the gene expression with messenger ribonucleic acid (mRNA) analysis.

Methods: CIC samples were obtained from thirty (n=30) patients with various OS diseases and ten (n=10) normal subjects; Schirmer test, Tear break-up time and OSDI score were also performed. Sample collection of CIC specimens was performed using EP device. It is carried out by applying the polyethesulfone filter adapted in the device onto the anaesthetized bulbar conjunctiva for a few seconds, after which it was removed. Cells were harvested from CIC samples and subjected to FC (FC500, Beckman Coulter) using monoclonal antibodies directed to HLA-DR, and real-time PCR (Applied Biosystems) was performed in parallel allowing to quantitatively analyzing the protein expression and mRNA transcripts of HLA-DR respectively.

Results: Enough RNA materials were collected using EP device with an average of 1µg/CIC. All patients included had high HLA-DR levels compared to healthy volunteers with induction in HLA-DR transcripts and protein expression. Positive correlation was found between mRNA and protein expression of HLA-DR (R square = 0.57 Pearson r = 0.75), and significant correlation (p<0.0001) was observed for the whole dataset.

Conclusions: This study showed an overall positive correlation between mRNA and protein expression assessed with FC. Moreover, the quantity of RNA material collected with the EYEPRIM device allows further gene expression investigation. This device appears useful for the impression cytology and molecular markers investigation. Finally, these results, since HLA-DR is a marker of OS inflammation, suggest that the relationship and correlation between protein and transcripts of other ocular diseases biomarkers need to be explored.

Keywords: 474 conjunctiva • 468 clinical research methodology