Sample acquisition.

Purpose
VitreoDx is an experimental device enabling push-button collection of a neat vitreous liquid biopsy incidental to an intravitreal injection. We explored the ability of the device to collect a sample usable for proteomic biomarker discovery and testing.
Design
Pilot study using ex vivo human eyes.
Methods
Non-vitrectomized, human eyes from nine donors 75–91 years of age were refrigerated in BSS and used within 5 days of death. Four VitreoDx devices fitted with 25G needles, and four staked needle insulin syringes with 30G needles, were inserted at equal intervals through the pars plana of each eye and held in place by a fixture. The sampling mode of each VitreoDx device was triggered to attempt to acquire a liquid biopsy up to 70 μL. The plunger of each insulin syringe was retracted to attempt to obtain a liquid biopsy with a maximum volume of 50 μL. Samples acquired with the VitreoDx were extracted to polypropylene cryovials, refrigerated to -80 ºC, and sent for offsite proteomic analysis by proximity extension assay with a focus on panels containing approved and pipelined drug targets for neovascular disease and inflammatory factors.
Results
Of the attempted liquid biopsies with the novel 25G VitreoDx, 92% (66 of 72) resulted in successful acquisition (>25 μL) while 89% (64 of 72) attempted by a traditional 30G needle resulted in a successful acquisition. Sample volume sufficient for proteomics array analysis was acquired by the VitreoDx for every eye. Detectable protein was found for 151 of 166 unique proteins assayed in at least 25% of eyes sampled by VitreoDx.
Conclusions
The high acquisition rate achieved by the prototype was similar to that achieved in previous clinical studies where a standard syringe was used with a 25G needle to biopsy vitreous fluid directly prior to standard intravitreal injection. Successful aspiration rates were likewise high for 30G needles. Together, these suggest that it is possible to routinely acquire liquid vitreous biopsies from patients who typically receive intravitreal injections with an injection device using a standard size needle without a vitreous cutter. Protein analysis shows that proteins of interest survive the sampling mechanism and may have potential to direct care in the future.

研究目的：VitreoDx是一款实验性装置，可在玻璃体内注射（intravitreal injection）过程中，通过一键操作便捷采集纯净的玻璃体液体活检（vitreous liquid biopsy）样本。本研究旨在评估该装置采集可用于蛋白质组学生物标志物发现与检测样本的能力。
研究设计：采用离体人眼（ex vivo human eyes）开展的先导性研究。
研究方法：招募9名年龄介于75~91岁的供体，获取其未行玻璃体切除术的人眼，将眼球置于平衡盐溶液（BSS）中冷藏，并于供体死亡后5天内完成实验操作。将4台配备25G针头的VitreoDx装置，与4台配备30G固定针的胰岛素注射器，以等间距方式经每只眼球的睫状体平坦部（pars plana）穿刺植入，并通过固定装置保持位置稳定。触发每台VitreoDx装置的采样模式，尝试采集最大体积达70μL的液体活检样本；回抽每支胰岛素注射器的活塞，尝试采集最大体积为50μL的液体活检样本。将VitreoDx装置采集到的样本转移至聚丙烯冷冻管（polypropylene cryovials）中，冷藏于-80℃环境，并送至外部实验室通过邻近延伸分析法（proximity extension assay）开展蛋白质组学分析，分析重点为包含新生血管性疾病（neovascular disease）获批药物靶点、在研药物靶点以及炎症因子的检测面板。
研究结果：采用新型25G VitreoDx装置完成的液体活检尝试中，92%（72次尝试中的66次）成功采集到体积大于25μL的样本；而采用传统30G针头完成的活检尝试中，89%（72次尝试中的64次）取得成功。VitreoDx装置可从每只眼球采集到满足蛋白质组芯片分析所需的样本体积。在通过VitreoDx装置采样的眼球中，对166种独特蛋白质进行检测后，有151种蛋白质可在至少25%的采样眼球中被检出。
研究结论：该原型装置实现的高采样成功率，与此前临床研究中采用标准注射器搭配25G针头，在标准玻璃体内注射前直接采集玻璃体液体活检样本的成功率相近；30G针头的成功抽吸率同样较高。综上，对于常规接受玻璃体内注射的患者，无需使用玻璃体切割机，仅需采用标准规格针头的注射装置即可常规完成玻璃体液体活检。蛋白质分析结果显示，目标蛋白质可在采样过程中保持活性，未来或可用于指导临床诊疗。