July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
Optimization and validation of a novel technique for in vivo imaging of FITC-dextran labelled erythrocytes in non-human primates
Author Affiliations & Notes
  • Elise L. Ma
    University of Maryland School of Medicine, Baltimore, Maryland, United States
  • Lakyn Mayo
    University of Maryland School of Medicine, Baltimore, Maryland, United States
  • Xiaoxuan Fan
    University of Maryland School of Medicine, Baltimore, Maryland, United States
  • Karen Underwood
    University of Maryland School of Medicine, Baltimore, Maryland, United States
  • Steven L Bernstein
    University of Maryland School of Medicine, Baltimore, Maryland, United States
  • Osamah Saeedi
    University of Maryland School of Medicine, Baltimore, Maryland, United States
  • Footnotes
    Commercial Relationships   Elise Ma, None; Lakyn Mayo, None; Xiaoxuan Fan, None; Karen Underwood, None; Steven Bernstein, None; Osamah Saeedi, Heidelberg Engineering (F), Heidelberg Engineering (R), Vasoptic Medical Inc (F)
  • Footnotes
    Support  NEI K23EY025014
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 189. doi:
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    • Get Citation

      Elise L. Ma, Lakyn Mayo, Xiaoxuan Fan, Karen Underwood, Steven L Bernstein, Osamah Saeedi; Optimization and validation of a novel technique for in vivo imaging of FITC-dextran labelled erythrocytes in non-human primates. Invest. Ophthalmol. Vis. Sci. 2019;60(9):189.

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      © ARVO (1962-2015); The Authors (2016-present)

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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.

 

FACS analysis in live monkey erythrocytes.

FACS analysis in live monkey erythrocytes.

 

FITC-labelled erythrocytes (arrows) in monkey fundus.

FITC-labelled erythrocytes (arrows) in monkey fundus.

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