Selective uptake of indocyanine green by reticulocytes in circulation.

Indocyanine green (ICG) is a fluorescent dye commonly used in ophthalmology to image retinal and choroidal vasculature. It is also used clinically to measure cardiac output, liver function, and peripheral circulation. Its absorption and emission in the near infrared region allow deep tissue penetration, making ICG angiography particularly useful for imaging choroidal circulation and neovascularization in ophthalmology.1,2 ICG has provided beneficial information toward identification and treatment regimens in various conditions that manifest retinal or choroidal neovascularization, hemorrhage, or vascular leakage. It has been particularly useful in the management of age-related macular degeneration and idiopathic polypoidal choroidal vasculopathy.3 ICG binds to blood plasma proteins immediately after injection and is cleared from the circulation in approximately 20 minutes.4,5 Recently, Matsuda et al.6 reported observation of ICG-labeled cells in retinal and choroidal vessels after dye clearance from the plasma. Based on their size and number, the ICG-labeled cells were thought to be circulating leukocytes. Given the involvement of leukocytes in retinal diseases such as diabetic retinopathy7 and retinal ischemia,8 it is important to determine whether leukocyte trafficking can indeed be monitored in vivo by a nontoxic dye such as ICG. Current fluorescent markers for leukocytes, such as acridine orange (AO) and rhodamine-6G (R6G),9,10 have significant side effects that affect their use in tracking circulating leukocytes. Both AO and R6G have been demonstrated to be mutagenic or carcinogenic,11–14 and both may have phototoxic effects.15–18 Consequently, neither is approved for clinical use. Therefore, it is important to investigate further the use of the nontoxic, clinically approved ICG, as an in vivo circulatory cell stain. Because image resolution is limited by the optics of the eye, the ICG-labeled cells in the report of Matsuda et al.6 (imaged with a scanning laser ophthalmoscope) appear as hyperfluorescent dots that are not well resolved. In contrast, in vivo confocal microscopy allows cellular imaging at much higher resolution in the skin (∼1-μm transverse and 3–5-μm axial resolution).19 Here, we used a real-time fluorescence confocal microscope to image cellular uptake of the ICG dye in the dermal microcirculation. In vivo double-labeling experiments using ICG and R6G reveal that two mutually exclusive cell populations take up these dyes. Furthermore, leukocytes labeled with R6G spontaneously roll on dermal vascular endothelium in vivo, whereas ICG-labeled cells do not interact with the dermal endothelium. To identify the ICG-stained cell population, we used a panel of antibodies against the following cell surface markers: CD45, CD71, CD16, and glycophorin A. CD45 is the leukocyte common antigen that is expressed on all hematopoietic cells not of erythroid lineage. It therefore is present on all leukocytes. Conversely, glycophorin A is expressed exclusively on cells of the erythroid lineage, regardless of developmental stage, whereas CD71 (transferrin receptor) functions in the uptake of iron in immature cells of the erythroid lineage.20–22 Mature erythrocytes are positive for glycophorin A (glycophorin A+), but negative for CD45 and CD71 (CD45− and CD71−, respectively). Reticulocytes (immature erythrocytes in the circulation), are CD45−, glycophorin A+, and CD71+. We identified the ICG-labeled cells as reticulocytes based on CD45−, glycophorin A+, and CD71+ immunostaining. In addition, there is a second cell population with distinctly weaker ICG staining that is CD45+ and CD16+. CD16 is a component of the low-affinity Fc receptor that functions in phagocytosis. Fc is the constant region common to all antibodies of a specific class. It was named “crystallizable fragment” because of the discovery of that property in IgG molecules. Unlike the variable antibody regions that recognize and bind to specific antigens, the Fc or constant region is recognized and binds to receptors on phagocytic cells of the immune system such as monocytes, macrophages, and granulocytes. Fc binding to phagocytic cells is critical to the destruction of foreign substance coated by antibodies. We identified these cells as neutrophils based on their CD45+ and CD16+ phenotypes. These weakly labeled cells most likely would not be detected in vivo. To our knowledge, this is the first report of selective and strong reticulocyte staining by ICG.