Cai Chengzhong, Carey Kai A, Nedosekin Dmitry A, Menyaev Yulian A, Sarimollaoglu Mustafa, Galanzha Ekaterina I, Stumhofer Jason S, Zharov Vladimir P
Arkansas Nanomedicine Center, University of Arkansas for Medical Sciences, Little Rock, Arkansas, 72205.
Division of Biochemical Toxicology, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, Arkansas, 72079.
Cytometry A. 2016 Jun;89(6):531-42. doi: 10.1002/cyto.a.22854. Epub 2016 Apr 14.
In vivo photoacoustic (PA) flow cytometry (PAFC) has already demonstrated a great potential for the diagnosis of deadly diseases through ultrasensitive detection of rare disease-associated circulating markers in whole blood volume. Here, we demonstrate the first application of this powerful technique for early diagnosis of malaria through label-free detection of malaria parasite-produced hemozoin in infected red blood cells (iRBCs) as high-contrast PA agent. The existing malaria tests using blood smears can detect the disease at 0.001-0.1% of parasitemia. On the contrary, linear PAFC showed a potential for noninvasive malaria diagnosis at an extremely low level of parasitemia of 0.0000001%, which is ∼10(3) times better than the existing tests. Multicolor time-of-flight PAFC with high-pulse repetition rate lasers at wavelengths of 532, 671, and 820 nm demonstrated rapid spectral and spatial identification and quantitative enumeration of individual iRBCs. Integration of PAFC with fluorescence flow cytometry (FFC) provided real-time simultaneous detection of single iRBCs and parasites expressing green fluorescence proteins, respectively. A combination of linear and nonlinear nanobubble-based multicolor PAFC showed capability to real-time control therapy efficiency by counting of iRBCs before, during, and after treatment. Our results suggest that high-sensitivity, high-resolution ultrafast PAFC-FFC platform represents a powerful research tool to provide the insight on malaria progression through dynamic study of parasite-cell interactions directly in bloodstream, whereas portable hand-worn PAFC device could be broadly used in humans for early malaria diagnosis. © 2016 International Society for Advancement of Cytometry.
体内光声（PA）流式细胞术（PAFC）已通过对全血中与罕见疾病相关的循环标志物进行超灵敏检测，在致命疾病的诊断方面展现出巨大潜力。在此，我们展示了这项强大技术在疟疾早期诊断中的首次应用，即通过无标记检测感染红细胞（iRBCs）中疟原虫产生的疟色素作为高对比度PA剂。现有的使用血涂片的疟疾检测方法能够在疟原虫血症为0.001 - 0.1%时检测出疾病。相反，线性PAFC显示出在极低疟原虫血症水平（0.0000001%）下进行无创疟疾诊断的潜力，这比现有检测方法好约10³倍。采用波长为532、671和820 nm的高脉冲重复率激光的多色飞行时间PAFC展示了对单个iRBCs的快速光谱和空间识别以及定量计数。将PAFC与荧光流式细胞术（FFC）相结合，分别实现了对单个iRBCs和表达绿色荧光蛋白的寄生虫的实时同步检测。基于线性和非线性纳米气泡的多色PAFC组合显示出通过在治疗前、治疗期间和治疗后对iRBCs进行计数来实时控制治疗效果的能力。我们的结果表明，高灵敏度、高分辨率的超快PAFC - FFC平台是一种强大的研究工具，可通过直接在血流中动态研究寄生虫 - 细胞相互作用来深入了解疟疾进展，而便携式可穿戴PAFC设备可广泛应用于人类疟疾的早期诊断。© 2016国际细胞计量学促进协会。
