Intravenous indocyanine green dye is insufficient for robust immune cell labelling in the human retina

It is not currently possible to reliably visualise and track immune cells in the human central nervous system or eye. Previous work demonstrated that indocyanine green (ICG) dye could label immune cells and be imaged after a delay during disease in the mouse retina. We report a pilot study investigating if ICG can similarly label immune cells within the human retina. Twelve adult participants receiving ICG angiography as part of routine standard of care were recruited. Baseline retinal images were obtained prior to ICG administration then repeated over a period ranging from 2 hours to 9 days. Matched peripheral blood samples were obtained to examine systemic immune cell labelling and activation from ICG by flow cytometry with human macrophage cultures as positive controls. Differences between the delayed near infrared ICG imaging and 488 nm autofluorescence was observed across pathologies, likely arising from the retinal pigment epithelium (RPE). Only one subject demonstrated ICG signal on peripheral blood myeloid cells and only three distinct cell-sized signals appeared over time within the retina of three participants. No significant increase in immune cell activation markers were detected after ICG administration. ICG accumulated in the endosomes of macrophage cultures and was detectable above a minimum concentration, suggesting cell labelling is possible. ICG can label RPE and may be used as an additional biomarker for RPE health across a range of retinal disorders. Standard clinical doses of intravenous ICG do not lead to robust immune cell labelling in human blood or retina and further optimisation in dose and route are required.目前尚无法在人类中枢神经系统或眼部实现免疫细胞的稳定可视化与追踪。既往研究证实，吲哚菁绿（indocyanine green, ICG）染料能够标记免疫细胞，且在小鼠视网膜疾病模型中可实现延迟成像。本研究开展一项先导试验，旨在探究ICG是否同样可标记人类视网膜内的免疫细胞。本研究招募了12名接受ICG血管造影（作为常规临床诊疗的一部分）的成年受试者。在注射ICG前获取基线视网膜图像，随后在2小时至9天的周期内重复采集图像。采集匹配的外周血样本，通过流式细胞术检测ICG诱导的全身免疫细胞标记与活化情况，并以人巨噬细胞培养物作为阳性对照。不同病理状态下均观察到延迟近红外ICG成像与488nm自发荧光之间存在差异，该差异可能源自视网膜色素上皮（retinal pigment epithelium, RPE）。仅1名受试者的外周血髓系细胞检测到ICG信号，且3名受试者的视网膜内仅在随访期间出现3处明确的细胞大小级信号。注射ICG后未检测到免疫细胞活化标志物的显著升高。ICG可在巨噬细胞培养物的内体中蓄积，且在最低检测浓度以上即可被检出，提示免疫细胞标记具备可行性。ICG可标记视网膜色素上皮（RPE），或可作为多种视网膜疾病中评估RPE健康状态的补充生物标志物。常规临床剂量的静脉注射ICG无法在人类血液或视网膜中实现稳定的免疫细胞标记，因此需要进一步优化给药剂量与给药途径。