Abstract
Purpose :
Sub-cellular structures within the vitreous, including exosomes and microparticles may be relevant to disease pathogenesis. Flow cytometry allows for single-particle (singlet) analysis, however particles adherent to one another (doublets or triplets) within the specimen hamper accurate fluorescent data collection and analysis. We used fresh human vitreous to identify effects of vitrectomy cut-rate, underlying retinal pathology, and delay time in sample analysis on retrieval of singlets using flow cytometry.
Methods :
Nine fresh human vitreous samples from patients undergoing vitrectomy for various vitreoretinal disorders were collected at different cut rates. Specimens were placed on ice and within 8 hours, incubated in a shaker with type II collagenase and cell culture media for 4 hours at 37°C. Specimens were spun down, washed, and resuspended in solution containing 4′,6-diamidino-2-phenylindole (DAPI) to evaluate microparticle membrane integrity. Specimens were analyzed using a BD FACS Canto II Flow Cytometer. SPSS Version 20 was used to perform Mann-Whitney U and Wilcoxon-Rank Sum tests.
Results :
Vitrectomy cut rates ranged from 1,000 cuts per minute (cpm) to 8,000 cpm. A lower cut rate (1,000 cpm) had greater singlet population per sample than higher cut rates (>4000 cpm) 87.7% ± 8.6 (n=4) (mean ± S.D.) vs 65.9% ± 12.8 (n=5), respectively, p = 0.016. Patients undergoing vitrectomy for complications from diabetes including non-clearing vitreous hemorrhage and tractional retinal detachment also had greater singlet populations than epiretinal membranes (ERM) or macular holes (MH), 87.7% ± 8.6 (n=6) vs 57.5% ± 7.1 (n=3), respectively, p=0.024. Prepared specimens with prolonged overnight incubation at 4°C prior to flow analysis showed a trend for greater uptake of DAPI fluorescence compared to those analyzed the day of specimen collection, p=0.109.
Conclusions :
Obtaining singlet populations of human vitreous microparticles is important for their accurate analysis. Lower cut rates may increase singlet-microparticle yield. Reduced singlet-populations in patients with ERM or MH may suggest a more viscous vitreous in these disease processes. Increased uptake of DAPI over 1 day may suggest breakdown of microparticle lipid bilayers demonstrating their relative pliability.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.