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A. Corell, H. Martinez-Osorio, M. Calonge, E. Gutierrez-San Jose, A. Lopez-Garcia, I. Fernandez, Y. Diebold; Flow Cytometry Characterization of Conjunctival Cells Obtained by Brush Cytology. Invest. Ophthalmol. Vis. Sci. 2008;49(13):6048. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To characterize by flow cytometry human conjunctival cells recovered by brush cytology (BC)
BC was performed in superior bulbar and tarsal conjunctivas of 20 healthy donors (Female: 13; Men: 7; age: 74.6 ± 1.8 yr) after performing an impression cytology. Cells were detached by shaking the brush in supplemented DMEM/F-12 medium + 10% FBS. This procedure was repeated three times. Recovered cells were counted and their initial viability was assessed by Trypan Blue. To characterize recovered cells, three flow cytometry assays were done and bulbar (n=10) vs tarsal (n=10) samples were compared in order to: 1) establish their lineage (An anti-cytokeratin [CK-7]-FITC MoAb was used as epithelial marker and an anti-CD45-PC7 MoAb as panleukocytic marker); 2) analyze the cell-cycle (propidium iodide (PI); and 3) assess the viability and apoptotic stage (Annexin V and PI). Two-tailed Student’s t test was performed for statistical analysis
Recovered cell numbers were 14 x 104 ± 0.9 cells (initial mean viability: 30.3 ± 1.1%). No significant differences were observed between bulbar and tarsal conjunctiva in terms of cell amount or viability, although more cells were recovered from bulbar area. CK-7+ cells represented the 67.8 ± 1.6% in tarsal BCs and the 65.7 ± 2.5% of bulbar BCs. However, bulbar cells showed higher density of CK7 (mean: 7.5 ± 0.8) than tarsal epithelium (mean: 4.6 ± 0.3) (p = 0.005). CD45+ cells were the 3.8 ± 0.4% of the tarsal cells and the 2.8 ± 0.3% of the bulbar cells. Regarding the DNA content, tarsal epithelial cells had higher proliferative index (S phase) than bulbar conjunctival cells (3.5 ± 0.3% vs. 2 ± 0.2%) (p = 0.0006). This means that close to 10% of the recovered cells were dividing, while more than 80% were either quiescent (G0) or pre-cycling (G1). Although the % of viable, apoptotic and dead cells showed no significant differences between tarsal and bulbar BCs, we observed two different populations: 22.8 % (± 1.6) of cells were smaller, less complex and had good viability (77.9 ± 3.4%); 76.4% (± 1.6) of cells were larger, more complex, and showed poor viability (21.3 ± 2.2%)
In healthy subjects, conjunctival cell populations recovered by BC are not purely epithelial, as there are a few CD45+ cells, not previously described. Additionally, the differences observed in terms of cell size, viability, and proliferative capacity of recovered cells may help to discriminate cell populations more suitable for the establishment of long-term cultures
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