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Hernan Martinez-Osorio, Nuria Nieto-Nicolau, Ricardo Pedro Casaroli-Marano, Rafael I Barraquer; Repeatability over time of several inflammatory biomarkers analyzed by flow cytometry in conjunctival brush cytology samples. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2005.
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Standard operative procedures are being established in several non-invasive techniques, as tear cytokine analysis, in order to incorporate into clinical trials. Sample collection, storage and shipping of conjunctival cells obtained by brush cytology (BC) was previously validated (Martínez-Osorio H, Calonge M, Corell A, Reinoso R, López A, Fernández I, San José EG, Diebold Y. Characterization and short-term culture of cells recovered from human conjunctival epithelium by minimally invasive means. Mol Vis. 2009 Oct 27;15:2185-95). In multicenter clinical trials, the samples are shipped for distant cities to travel up to 48 hours. To overcome this limitation, we established the repeatability for up to 3 days of Multitest CD3/CD4/CD38/HLA-DR biomarkers analyzed by flow cytometry (FC) in conjunctival cells obtained by BC
Inferior bulbar conjunctival cells were obtained by BC as previously published. Samples were shipped and storage at room temperature (15-20 degreesC) in SHEM medium (3:1). Multitest CD3/CD4/CD38/HLA-DR commercial kit was used. Isotype-matched unspecific monoclonal antibodies were used as negative controls. FC analysis was performed with a Gallios flow cytometer (Beckman-Coulter). The percentage of lived BC-recovered conjunctival cells was determined by staining with DAPI. Percentage of lived, CD3, CD4, CD38 and HLA-DR cells was assessed on day 1, 2 and 3
Cellular viability of epithelial cells at day 1, 2 and 3 was 18%. HLA-DR expression was less than 4% in healthy samples. 2% of BC cells were CD3 positive and 58% of CD3 cells were CD4 positive. These percentages have not changed for up to 3 days
The viability and inflammatory expression of conjunctival cells obtained by BC were unchanged for up to 3 days at room temperature. The routine use of this combined technique, BC and FC, in clinical trials may provide objective biomarkers for the diagnosis, treatment and to study the natural course of the ocular surface diseases
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