The 1-Step Versus 2-Step Subretinal Injection Trial (1,2-SIT)-A Randomized Controlled Trial to Compare Drug Reflux Following Subretinal Injection.

Purpose: To estimate in humans, in vivo, drug retention in the subretinal space following either 1- or 2-step subretinal injection (SRI).

Design: A single-masked, randomized, controlled trial.

Methods: Patients presenting with submacular hemorrhage secondary to age-related macular degeneration were randomly allocated to receive subretinal tissue plasminogen activator (50 µg in 0.1 mL) with sodium fluorescein (10 µg in 0.1 mL) as an optical label either as a 1-step (n = 6) procedure, in which the drug defines the subretinal space, or as a 2-step (n = 6) procedure, in which balanced salt solution is first used to define the subretinal space, following pars plana vitrectomy. All patients underwent air-for-fluid exchange at the completion of surgery with subsequent 20% sulfahexafluoride gas and bevacizumab injection. Reflux of subretinally injected drug was calculated by performing fluorophotometry on the fluid collected at the end of air-for-fluid exchange. Patients received intravitreal anti-VEGF at 4-weekly intervals to the final follow-up at 12 weeks. The primary outcome measure was the proportion of drug reflux. Secondary outcomes included duration of surgery, change in visual acuity (VA), final VA, final foveal thickness, and change in foveal thickness. To determine our fluorophotometric technique's applicability to gene and cell therapy, real-time quantitative polymerase chain reaction was employed to determine adeno-associated viral (AAV) yields following exposure to 0.1 mg/mL sodium fluorescein and its effects on retinal progenitor cells (RPCs) was assessed using a cell viability assay.

Results: Mean reflux was 4.8% ± 3.1% (mean ± SEM, range 0.4%-19.5%) for 1-step SRI and 3.9% ± 0.9% (range 1.7%-5.3%) for 2-step SRI (no significant difference in means; P = .0155 for the difference in variance). There was no significant difference in the duration of surgery (26.8 ± 1.2 minutes vs 30 ± 2.7 minutes), final VA (1.1 ± 0.26 [Snellen 20/252] vs 1.1 ± 0.32 [Snellen 20/252] logMAR), change in BCVA (-0.45 ± 0.27 vs -0.27 ± 0.23 logMAR) or foveal thickness (139.2 ± 33.2 µm vs 129.8 ± 21.1 µm). Quantitative polymerase chain reaction confirmed that AAV titers are not affected by 0.1 mg/mL sodium fluorescein in vitro, and viability assays suggest that it does not adversely affect RPC viability.

Conclusions: This study demonstrates that drug loss following SRI ranged from 0.4% to 19.8% (mean 4.3%). There is no significant difference between 1-step and 2-step SRI in the mean proportion of drug reflux, duration of surgery, change in neural retinal thickness, or change in BCVA. However, there is a significantly greater variability in reflux for 1-step injection compared to 2-step injection. AAV yields are not affected by 0.1 mg/mL sodium fluorescein, nor is RPC viability. These data suggest that sodium fluorescein may be an appropriate means of tracking subretinal AAV gene therapy and retinal cell therapy quantitatively and that the 2-step SRI approach is preferable to 1-step SRI to ensure consistency in drug delivery.