Abstract
In vivo photoacoustic flow cytometry (PAFC) has demonstrated potential for early diagnosis of deadly diseases through detection of rare circulating tumor cells, pathogens, and clots in nearly the entire blood volume. Before clinical application, this promising diagnostic platform requires verification and optimization using adequate preclinical models. We show here that this can be addressed by examination of large mouse blood vessels which are similar in size, depth and flow velocity to human vessels used in PAFC. Using this model, we verified the capability of PAFC for ultrasensitive, noninvasive, label-free, rapid malaria diagnosis. The time-resolved detection of delayed PA signals from deep vessels provided complete elimination of background from strongly pigmented skin. We discovered that PAFC's sensitivity is higher during examination of infected cells in arteries compared to veins at similar flow rate. Our advanced PAFC platform integrating a 1060 nm laser with tunable pulse rate and width, a wearable probe with a focused transducer, and linear and nonlinear nanobubble-amplified signal processing demonstrated detection of parasitemia at the unprecedented level of 0.00000001% within 20 seconds and the potential to further improve the sensitivity 100-fold in humans, that is approximately 106 times better than in existing malaria tests.
Highlights
Conventional flow cytometry using scattering and fluorescent detection methods has been for many years a fundamental tool of discoveries in cell biology and disease diagnosis [1]
In vivo photoacoustic (PA) flow cytometry (PAFC) is based on the irradiation of circulating targets with short laser pulses followed by timeresolved detection of laser-induced acoustic waves with an ultrasound transducer gently placed on the skin [3,4,5]
Most mouse vessels are much smaller than respective superficial human vessels for photoacoustic flow cytometry (PAFC) detection [3,4,22]
Summary
Conventional flow cytometry using scattering and fluorescent detection methods has been for many years a fundamental tool of discoveries in cell biology and disease diagnosis [1]. Invasive extraction of cells from a living organism, may lead to changes in cell properties, prevent the long-term study of cells in their native environment, limit sensitivity due to a small blood sample volume, and require multiple time-consuming sampling. Most of these problems can be solved by the use of in vivo flow cytometry, which provides noninvasive, continuous examination of nearly the entire blood volume circulating in the peripheral blood vessels [2]. A PAFC clinical prototype with hand-worn PA probe, demonstrated detection of CTCs in 1-2 mm blood vessels at depth of 1-3 mm with the sensitivity of 100 CTC/mL in melanoma patients [6] that was approximately 100-fold better than that seen with existing CTC assays ex vivo [7]
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