Powless Amy J, Prieto Sandra P, Gramling Madison R, Chen Jingyi, Muldoon Timothy J
Biomedical Engineering Department, University of Arkansas, Fayetteville, AR 72701, USA.
Pat Walker Health Center, University of Arkansas, Fayetteville, AR 72701, USA.
Diagnostics (Basel). 2020 Dec 12;10(12):1082. doi: 10.3390/diagnostics10121082.
Low-cost imaging systems that utilize exogenous fluorescent dyes, such as acridine orange (AO), have recently been developed for use as point-of-care (POC) blood analyzers. AO-based fluorescence imaging exploits variations in emission wavelength within different cell types to enumerate and classify leukocyte subpopulations from whole blood specimens. This approach to leukocyte classification relies on accurate and reproducible colorimetric features, which have previously been demonstrated to be highly dependent on the cell staining protocols (such as specific AO concentration, timing, and pH). We have developed a light-sheet-based fluorescence imaging spectrometer, featuring a spectral resolution of 9 nm, with an automated spectral extraction algorithm as an investigative tool to study the spectral features from AO-stained leukocytes. Whole blood specimens were collected from human subjects, stained with AO using POC methods, and leukocyte spectra were acquired on a cell-by-cell basis. The post-processing method involves three steps: image segmentation to isolate individual cells in each spectral image; image quality control to exclude cells with low emission intensity, out-of-focus cells, and cellular debris; and the extraction of spectra for each cell. An increase in AO concentration was determined to contribute to the red-shift in AO-fluorescence, while varied pH values did not cause a change in fluorescence. In relation to the spectra of AO-stained leukocytes, there were corresponding red-shift trends associated with dye accumulation within acidic vesicles and at increasing incubation periods. The system presented here could guide future development of POC systems reliant on AO fluorescence and colorimetric features to identify leukocyte subpopulations in whole blood specimens.
最近开发了利用吖啶橙（AO）等外源性荧光染料的低成本成像系统，用作即时护理（POC）血液分析仪。基于AO的荧光成像利用不同细胞类型内发射波长的变化来枚举和分类全血标本中的白细胞亚群。这种白细胞分类方法依赖于准确且可重复的比色特征，此前已证明这些特征高度依赖于细胞染色方案（如特定的AO浓度、时间和pH值）。我们开发了一种基于光片的荧光成像光谱仪，光谱分辨率为9nm，具有自动光谱提取算法作为研究工具，以研究AO染色白细胞的光谱特征。从人类受试者采集全血标本，使用POC方法用AO染色，并逐个细胞获取白细胞光谱。后处理方法包括三个步骤：图像分割以在每个光谱图像中分离单个细胞；图像质量控制以排除发射强度低的细胞、失焦细胞和细胞碎片；以及提取每个细胞的光谱。已确定AO浓度的增加会导致AO荧光的红移，而不同的pH值不会引起荧光变化。关于AO染色白细胞的光谱，在酸性囊泡内和孵育时间增加时，染料积累存在相应的红移趋势。本文介绍的系统可为未来依赖AO荧光和比色特征来识别全血标本中白细胞亚群的POC系统的开发提供指导。
