Purchase this article with an account.
Pierre Roy, Hervé Groux, Francoise Cottrez, Laurene Protat; Assessment of the Eyeprim® Device for Conjunctival Impression and PCR. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5444.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
To evaluate the use of Conjunctival impression (CI) EYEPRIM device, that previously demonstrated increased cell collection and reproducibility than the current CI method, for the measurement of 3 biomarkers HLA-DR, CCR4, CCR5 of the ocular surface by quantitative Polymerase Chain Reaction (qPCR) following different extraction methods.
CI specimens of the upper bulbar conjunctiva were collected with EYEPRIM from 4 healthy volunteers in right eye (RE) and left eye (LE), without anaesthesia. Specimens were transferred in tubes containing 1ml of different extraction media: Group 1 in Phenol/guanidine-based Qiazol Lysis Reagent (QIAGEN) vortexed 1min, Group 2 in Qiagen RLT soft buffer medium vortexed 1min and groups 3 & 4 in RLT buffer medium with steel beads (5mm dia.). Group 3 was agitated softly and group 4 transferred into the tissue lyser II (Qiagen) for 2 cycles of 1.5min. RE and LE of each volunteer were processed with the same extraction protocol to assess the reproducibility of the results. The mRNA was extracted using the RNeasy kit (Qiagen) and processed by qPCR with HLA-DR, CCR4 and CCR5 specific primers.
For all the extraction protocols, enough ARNm material was collected to process the 3 biomarkers. Group 1 QIAzol Lysis Reagent + vortex, gave the best results with an average of 300ng of ARNm collected, followed by Group 3. Group 2 was the least efficient method. Group 4 gave intermediate results. qPCR gave 4 different profiles of biomarkers for each volunteer: First volunteer had little HLA-DR, no CCR4 and no CCR5. second had low expression level of all 3 biomarkers. third had highest levels of HLA-DR and CCR5 compared with the others, last had only CCR5. None expressed significant levels of CCR4.
EYEPRIM is an effective sampling tool, compatible with biomarker analysis by qPCR. This feasibility study showed a very good reproducibility of the samples, and different profiles of conjunctiva, characterized by biomarkers, were found. Further clinical studies with characterized pathological patients should be completed to identify the pathological threshold of those biomarkers, in order to confirm the potential use of EYEPRIM for the diagnosis of ocular surface disorders. Special attention should be paid to the extraction protocol.
This PDF is available to Subscribers Only