Centre for Ocular Research and Education, University of Waterloo, School of Optometry & Vision Science, 200 University Avenue West, N2L 3G1, ON, Canada.
Centre for Ocular Research and Education, University of Waterloo, School of Optometry & Vision Science, 200 University Avenue West, N2L 3G1, ON, Canada; Centre for Eye and Vision Research (CEVR), 17W Hong Kong Science Park, Hong Kong.
Exp Eye Res. 2021 Jun;207:108607. doi: 10.1016/j.exer.2021.108607. Epub 2021 Apr 28.
The purpose of this study was to develop a standardized, accurate and efficient method for estimating conjunctival goblet cell density (GCD) via optimizing sample storage conditions and quantification methods. Conjunctival impression cytology (CIC) membranes were collected from both eyes of 32 participants and were randomized to two storage durations (2-3 weeks, 6-7 weeks) and two storage container types (microcentrifuge tube, flat histology cassette). The CIC membranes were stained and subdivided into 25 areas (5 mm × 5 mm) for imaging and the GCs were counted under 200X magnification using three different methods: (1) full CIC membrane GC count of the 25 images with cell-counting software ("full"; reference method), (2) partial membrane GC count of 9 images with cell-counting software ("partial"), and (3) manual counting of the 25 images ("manual"). In all cases, GCD was determined by dividing the GC count by the counting area. The average time required for quantification was recorded to gauge efficiency. Results showed no significant difference in GC count between the two storage durations (p = 0.745) or storage container types (p = 0.552). The median (interquartile range (IQR)) time required to quantify a CIC membrane for the full, partial, and manual methods of GC counting, was 14.8(17.6), 4.6(5.2) and 5.0 (5.0) minutes, respectively. The agreement of GCD values between the full and manual methods (bias: 0.4, 95% LOA: [-4.6, 5.5]) was stronger than that comparing the full and partial methods (bias: 0.5, 95% LOA: [-18, 17]). All together, through systematic examination of key procedural variables, an optimized method for GCD quantification within 7 weeks of sample collection was outlined. Adaption of procedures described in this paper to facilitate accurate and efficient GCD quantification may serve as a valuable step in clinical trials investigating DED pathophysiology and/or novel DED treatment strategies.
本研究旨在通过优化样本储存条件和定量方法，开发一种标准化、准确和高效的方法来评估结膜杯状细胞密度（GCD）。从 32 名参与者的双眼收集结膜印片细胞学（CIC）膜，并将其随机分为两种储存时间（2-3 周，6-7 周）和两种储存容器类型（微量离心管、平皿组织学盒）。对 CIC 膜进行染色，并将其分为 25 个区域（5mm×5mm）进行成像，并在 200X 放大倍数下使用三种不同方法对 GCs 进行计数：（1）使用细胞计数软件对 25 张图像的全 CIC 膜 GC 计数（“全”；参考方法），（2）使用细胞计数软件对 9 张膜的部分 GC 计数（“部分”），和（3）对 25 张图像进行手动计数（“手动”）。在所有情况下，GC 密度通过将 GC 计数除以计数面积来确定。记录定量所需的平均时间以衡量效率。结果显示，两种储存时间（p=0.745）或储存容器类型（p=0.552）之间的 GC 计数无显著差异。全、部分和手动 GC 计数方法对 CIC 膜进行定量所需的中位数（四分位距（IQR））时间分别为 14.8（17.6）、4.6（5.2）和 5.0（5.0）分钟。全和手动方法之间的 GCD 值一致性（偏差：0.4，95% LOA：[-4.6，5.5]）强于全和部分方法之间的一致性（偏差：0.5，95% LOA：[-18，17]）。总的来说，通过对关键程序变量的系统检查，提出了一种在样本采集后 7 周内进行 GCD 定量的优化方法。本研究中描述的程序的改编可能有助于准确和高效的 GCD 定量，这可能是研究 DED 病理生理学和/或新型 DED 治疗策略的临床试验中的一个有价值的步骤。
