Quantification of Diabetes-induced Adherent Leukocytes in Retinal Vasculature

Leukostasis refers to the attachment of leukocytes to the luminal wall of the vasculature. This interaction of leukocytes with the wall of blood vessels is characteristic of inflammation and has been causally linked to capillary occlusion in a variety of tissues and diseases, including diabetic retinopathy. Leukostasis has been reported for years as a life-threatening complication of hyperleukocytosis and can only be diagnosed clinically. Given the importance of the phenomenon, intensive research has been done to understand the potential mechanism(s) that lead to its manifestation; however, there is no gold-standard technique in laboratory settings to visualize and quantify the severity of the event. In the method summarized below, the vasculature is initially perfused with a buffer to remove blood, and then, concanavalin A is perfused into the vasculature where it binds to all exposed cell walls and causes especially bright staining of leukocytes. If the perfusion to remove all unbound blood cells was successful, the remaining fluorescently labeled leukocytes are bound to the vasculature, and they can be manually quantified using any available fluorescence microscope.