Validation of a Standardized Metabo-Lipidomic Workflow for Human Conjunctival Impression Cytology Specimens.

PURPOSE: The aim of this study was to validate a workflow for the metabo-lipidomic analysis of conjunctival impression cytology (CIC) specimens using ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry.

DESIGN

Validation study.

METHODS

Four CIC specimens were collected from both eyes of 20 healthy subjects, resulting in a total of 160 specimens. Three different metabolite extraction protocols (methanol, methanol/water, and acetonitrile), along with 2 lipid extraction protocols (methyl tertbutyl-ether and isopropanol) were assessed. The metabolites and metabolic pathways identified in conjunctival cells were compared with those found in human tears.

RESULTS

We identified 211 metabolites involved in 9 pathways. Although there was considerable variability in the 4 consecutive CIC specimens collected from the same eye, the first 2 CIC were found to be comparable. The conjunctival metabolome was found to be comparable between the right and left eyes. Comparison of the 137 metabolites identified in tears and CIC specimens revealed 79 common metabolites, while 132 were specific to CIC specimens and 58 were specific to tears. Isopropanol extraction was the method of choice for lipidomic, enabling the identification of 262 lipids from 24 lipid classes.

CONCLUSIONS

These results confirm the feasibility of metabo-lipidomic analysis using CIC specimens and demonstrate the comparability of the conjunctival metabolome between both eyes in healthy subjects. The use of successive CIC is a source of variability and the metabolome of conjunctival cells differs from that of tears. To facilitate biomarker research in clinical practice, we propose a standardized workflow for the metabo-lipidomic analysis of CIC specimens.