Abstract
Purpose :
Erythrocyte Mediated Angiography (EMA) is a technique that permits in vivo visualization of erythrocyte flow and dynamics at a microvasculature level. Use of indocyanine green EMA has shown impaired vasomotion in glaucoma and retinal vein occlusion (Flower&Kling, 2017). We theorized that an alternate method of EMA with fluorescein would offer several pre-clinical and clinical benefits; however, this method is hindered by the low molecular weight of fluorescein. In this study, we evaluated the capability of fluorescein EMA by encapsulating fluorescein isothiocynate-dextran (FITC-Dx) molecules within human erythrocytes, and validated the use of this method in a non-human primate (NHP).
Methods :
Primary human erythrocytes (RBCs) were isolated from whole blood and loaded with FITC-Dx at varying sizes (4K, 40K g/mol) and concentrations (100-2000uM) using a previously described osmotic shock technique. Optimal FITC-Dx uptake was determined by fluorescent signal level at 50ms exposure. To validate this method in vivo, blood was obtained by venipuncture from rhesus macaques, and isolated RBCs were loaded with FITC-Dx-40K (325uM) as above. Loading efficacy was quantified by flow cytometry using single-cell gating and emissions of 670nm (negative control) and 530nm (positive FITC). FITC-Dx loaded NHP RBCs were re-injected and real-time ocular blood flow was recorded with a Heidelberg fundus camera.
Results :
Human RBC in vitro trials showed optimized fluorescence uptake with FITC-Dx 40K at a concentration of 325uM, compared to other concentrations (100-300uM), or with the smaller FITC-Dx 4K at any tested concentration (vs 40K-other: p=0.015; vs 4K-all: p=0.0284; Kruskal-Wallis with multiple comparisons). FACS analysis of FITC-Dx 40K loaded NHP RBCs demonstrated 98.8% FITC-labelled singlets (positive) out of 33,745 events with 0% labelling at 670nm (negative control) [Fig.1]. Imaging of re-injected RBCs revealed in vivo visualization of FITC-Dx labelled cells in the monkey’s fundus [Fig. 2].
Conclusions :
FITC-Dx-labelled EMA may provide a novel technique for high-resolution, real-time imaging of retinal microvascular flow dynamics and the ability to simultaneously capture infrared images such as Ocular Coherence Topography using commercially-available fundus cameras.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.