Department of Biomedical Engineering, Texas A&M University, College Station, TX 77843, USA.
Department of Nutrition and Food Science, Texas A&M University, College Station, TX 77843, USA.
Anal Methods. 2021 May 20;13(19):2238-2247. doi: 10.1039/d1ay00195g.
Effective staining of peripheral blood smears by increasing contrast of intracellular components and biomarkers is essential for the accurate characterization, diagnosis, and monitoring of various diseases such as malaria. To assess the potential for automation of stained whole human blood smears at the point-of-care (POC), brightfield and fluorescence staining protocols were adapted for smears generated in channels of pumpless microchannels and compared to a standard glass smear. A 3× concentration Giemsa brightfield staining solutions (10, 33, and 50% dilution), and Acridine Orange fluorescence staining solutions (12 μg mL-1) were evaluated with human blood smears containing malaria parasites within a microfluidic channel. Giemsa staining at 33% dilution showed an optimal combination of contrast and preservation of cellular morphology, while 50% dilutions showed significant cellular crenation and 10% dilutions did not show desired contrast in brightfield imaging. Fluorescence staining at 12 μg mL-1 using Acridine Orange showed clear separability between the fluorescent intensities of the malaria parasites and that of the red blood cells (RBCs) and background. However, compared to glass smears, these exhibited reduced signal intensity as well as inverted contrast of RBCs and background. These results demonstrate that peripheral thin blood smears generated in pumpless microfluidic can be successfully stained in-channel with a simple, one-step procedure to permit brightfield and fluorescence imaging.
为了准确地描述、诊断和监测疟疾等各种疾病，通过增加细胞内成分和生物标志物的对比度来有效染色外周血涂片至关重要。为了评估在即时护理点（POC）对染色的全血涂片进行自动化处理的潜力，我们对无泵微通道中生成的涂片进行了明场和荧光染色方案的调整，并将其与标准玻璃涂片进行了比较。我们评估了 3×浓度的吉姆萨明场染色溶液（10%、33%和 50%稀释）和吖啶橙荧光染色溶液（12μg/mL），并在微流控通道中含有疟原虫的人类血涂片上进行了测试。33%稀释的吉姆萨染色显示出对比度和细胞形态保存的最佳组合，而 50%稀释则显示出明显的细胞皱缩，10%稀释则在明场成像中没有显示出所需的对比度。使用吖啶橙的荧光染色在 12μg/mL 时显示出疟原虫与红细胞（RBC）和背景之间荧光强度的清晰可分性。然而，与玻璃涂片相比，这些涂片的信号强度降低，并且 RBC 和背景的对比度颠倒。这些结果表明，无泵微流控中生成的外周薄血涂片可以通过简单的一步程序成功地在通道内进行染色，以允许进行明场和荧光成像。
