Jean-Fran$ois Le Gargasson Michel Paques Jean-Eric Guez Bernadette Boval Eric Vicaut Xin Hou Yvon Grail Alain Gaudric Scanning laser ophthalmoscope imaging of fluorescein-labelled blood cells Received: 9 April 1996 Revised version received: 22 July 1996 Accepted: 29 August 1996 Proprietary interest category: N J.F. Le Gargasson (~) • J.-E. Guez • Y. Grall Biophysics Department, H6pital Lariboisi~re, 2 rue Ambroise Part, F-75010 Paris, France Tel. +33-1-49 95 81 08; fax +33-1-49 95 81 15 M. Paques • A. Gaudric Ophthalmology Department, H6pital Lariboisi~re, Paris, France B. Boval Banque du Sang, H6pital Lariboisi~re, Paris, France E. Vicaut • X. Hou INSERM U141, H6pital, Fernand Widal, Paris, France Abstract • Purpose: To demon- strate the feasibility of a technique for the visualization by scanning laser ophthalmoscope (SLO) of flu- orescein-labelled autologous leuko- cytes and platelets in retinal ves- sels. • Method: Individual blood samples from rats and rabbits were centrifuged to isolate platelets and leukocytes, then passively labelled with fluorescein and reinjected into the same animal. An SLO was used to visualize and record cell dis- placement in the retinal circulation. Labelled platelets were analysed by flow cytometry. • Results: By SLO, platelets appeared as a het- erogeneous particle flow, and indi- vidual leukocytes appearing as brighter spots could easily be traced. Flow cytometry showed that after labelling platelets were well individualized and their size was slightly increased. • Conclusion: Circulating blood cells can be visu- alized in retinal vessels by a simple method consisting of passive la- belling of autologous platelets and leukocytes by fluorescein. No platelet toxicity was detected. This method could be applied to the study of blood cell movement in hu- man retinal vascular diseases. Introduction An important role for leukocytes in retinal vascular dis- eases such as diabetic retinopathy and venous thrombo- sis has recently been suggested [11]. Presently there is no method allowing precise analysis of blood cell circula- tion in human retinal vessels. At the experimental level, new methods have recently been developed for the visu- alization of circulating particles in the retinal vessels by scanning laser ophthalmoscopy (SLO). Koobehi and Peyman [7] used liposome-encapsulated dye (fluores- cein or indocyanine green) to measure the plasmatic flow in retinal, choroidal and optic nerve vessels. Nishiwaki et al. [10] and Kimura et al. [6] used intravenous acridine orange to label rat leukocytes. However, these methods are not yet suitable for human use. In the present study we describe a method of analyz- ing the retinal circulation of fluorescein-labelled au- tologous platelets and leukocytes by SLO which could theoretically be applied to human studies [8]. Materials and methods All experiments were performed in accordance with the Principles of Laboratory Animal Care of the NIH. Pigmented rabbits were used. Autologous platelet and leukocyte concentrates were pre- pared from the blood of each animal according to the protocol previously described [3, 5] with slight modifications. Briefly, a blood sample was taken from the ear of the rabbit, mixed with Acid Dextrose Formula A (ACDA) and treated under sterile conditions as follows: the blood was transferred into a tube and centrifuged for 15 min at 280 g at room temperature. The platelet-rich plasma devoid of red blood cells was decanted and diluted 1/8 v/v in ACDA before a second centrifugation for 10 rain at room tempera- ture and 1000 g. The supernatant platelet-poor plasma was drawn off, and the packed platelets were gently mixed with 0.6 ml of isotonic sodium chloride to obtain a platelet suspension free of agglutinates. The average concentration of this suspension was 109 platelets/ml. It was incubated in 10% sodium fluorescein for 25 miu. Excess fluorescein was washed out before intravenous injec- tion of the suspension. The animals were anaesthetized and their pupils dilated. Next, the fluorescein-labelled cells were injected into the ear vein of the rabbit. The circulating fluorescent platelets were then visualized by SLO. Images were recorded on video tapes.