Analysis of fluorescent images of respiratory epithelial cells from healthy donors

We applied automated image analysis to systematically examine the influence of samples’ storage conditions on the specificity of IF staining with eight polyclonal antibodies routinely used for diagnostics of primary ciliary dyskinesia, PCD. We used antibodies targeting epitopes of proteins representing various axoneme structural complexes. Seven various combinations of temperatures and storage times were tested to mimic the procedures of handling epithelial brushing on glass slides: at the clinic, during transport, or after reception at the diagnostic laboratory. Our study revealed that proper slide storage conditions are essential for the reliable PCD diagnosis via IF staining. Storage temperatures tested and their symbols used in the data files: Condition symbol Group Conditions (temperature and duration time) 1 -80_28d (best conditions) -80°C 28 days 3 -20_28d -20°C28 days 4 -20_8w -20°C 8 weeks 5 -20/RT/-80 -20°C 11 days RT 3 days -80°C 14 days 6 4/RT/-80 4 °C 11 days RT 3 days -80°C 14 days 7 RT/RT/-80 RT 11 days RT 3 days -80°C 14 days 2 RT_28d (worst conditions) RT 28 days我们采用自动化图像分析技术，系统探究了样本存储条件对免疫荧光（IF，Immunofluorescence）染色特异性的影响。本实验所用8种多克隆抗体均为原发性纤毛运动障碍（PCD，Primary Ciliary Dyskinesia）诊断的常规试剂，这些抗体靶向的蛋白表位涵盖多种轴丝结构复合物。我们设置了7组温度与存储时长的组合，以模拟玻璃玻片上皮刷检样本的实际处理流程，涵盖临床采样、运输及诊断实验室接收后的全环节。 本研究证实，规范的玻片存储条件对依托免疫荧光染色开展的原发性纤毛运动障碍可靠诊断至关重要。 本实验测试的存储温度及对应数据文件标识如下： 1. 标识：-80_28d（最佳条件）：-80℃，存储28天 3. 标识：-20_28d：-20℃，存储28天 4. 标识：-20_8w：-20℃，存储8周 5. 标识：-20/RT/-80（RT，Room Temperature，室温）：-20℃存储11天，室温存储3天，-80℃存储14天 6. 标识：4/RT/-80：4℃存储11天，室温存储3天，-80℃存储14天 7. 标识：RT/RT/-80：室温存储11天，室温存储3天，-80℃存储14天 2. 标识：RT_28d（最差条件）：室温存储28天