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Alterations of blood rheology (hemorheology) are important for the early diagnosis, prognosis, and prevention of many diseases, including myocardial infarction, stroke, sickle cell anemia, thromboembolism, trauma, inflammation, and malignancy. However, real-time in vivo assessment of multiple hemorheological parameters over long periods of time has not been reported. Here, we review the capabilities of label-free photoacoustic (PA) and photothermal (PT) flow cytometry for dynamic monitoring of hemorhelogical parameters in vivo which we refer to as photoacoustic and photothermal blood rheology. Using phenomenological models, we analyze correlations between both PT and PA signal characteristics in the dynamic modes and following determinants of blood rheology: red blood cell (RBC) aggregation, deformability, shape (e.g., as in sickle cells), intracellular hemoglobin distribution, individual cell velocity, hematocrit, and likely shear rate. We present ex vivo and in vivo experimental verifications involving high-speed PT imaging of RBCs, identification of sickle cells in a mouse model of human sickle cell disease and in vivo monitoring of complex hemorheological changes (e.g., RBC deformability, hematocrit and RBC aggregation). The multi-parameter platform that integrates PT, PA, and conventional optical techniques has potential for translation to clinical applications using safe, portable, laser-based medical devices for point-of-care screening of disease progression and therapy efficiency. © 2011 International Society for Advancement of Cytometry
Blood rheology (also termed hemorheology) is an integrated branch of physics and medicine that deals with the blood flow and deformation behavior of blood components (1, 2). Changes of blood rheology are thought to be crucial in the progression of many severe diseases, such as stroke, heart attack, anemia, diabetes, and sickle cell disease (1, 3–5). These changes may also dramatically complicate therapeutic interventions (e.g., infusion of heparin or warfarin). The grand challenge of hemorheology in live organism is the complexity of multiple dynamic relationships. The most important rheological property of blood is its resistance to flow, or viscosity. The major flow-affecting determinants of whole-blood viscosity, and thus hemorheology, are 1) hematocrit (Ht), the fraction of the blood volume occupied by red blood cells (RBCs); 2) RBC deformability, the ability of RBCs to undergo deformation in flow, depending on the shape, membrane deformability, and other properties (e.g., chemical structure or clustering capability) of hemoglobin (Hb); and 3) RBC aggregation, the formation of reversible (physiological) rouleaux or irreversible (pathological) clumps of RBCs. Viscosity is also determined by shear rates (velocity difference) and flux of RBCs (number of RBCs per second)(1–4). Another rheological determinant, plasma viscosity, usually has little influence on whole-blood viscosity. Indeed, when RBCs are added to plasma, blood viscosity becomes increasingly sensitive to Ht, with an almost exponential relationship between the Ht value and blood viscosity (1). In particular, at moderate-to-high shear rates, a 1% change in Ht (from 45% to 46%) increases blood viscosity by 4%.
Hemorheology has been intensively studied for decades by many methods ex vivo, including rotational, viscosimetry, micropipette aspiration, sedimentation rate, micropore filtration, and flow cytometry (1–5). However, these methods are limited by one or more of the following factors: 1) single-parameter measurement (e.g., RBC aggregation or Ht); 2) invasiveness, which may unpredictably alter rheological properties and prevent long-term monitoring in the native biological environment; 3) time-consuming preparation procedures (several hours if not an entire day); 4) discontinuous sampling with limited, discrete time points; and 5) the small blood volume extracted (typically a few milliliters). Furthermore, ex vivo/in vitro tests cannot replicate the transient flow-affecting character of rheological changes, which only become fully apparent in the natural blood circulation in vivo under the influence of endothelial, nervous, and humoral stimuli.
Technological progress in the development of methods for evaluating blood rheology in vivo promises to extend its clinical significance (2). Among numerous in vivo diagnostic techniques (e.g., ultrasound, magnetic resonance imaging, positron emission tomography, and various optical techniques), photothermal (PT), and photoacoustic (PA) methods have exhibited a high level of sensitivity in detecting of individual cells (6–10) and monitoring oxygenation and total Hb content in humans (11–13). Fluorescence techniques can also detect and image individual cells and blood flow (14–18). However the use of fluorescence labeling in vivo raises potential problems for translating this technology to humans because of1) the cytotoxicity of available fluorescent tags,2) photobleaching or blinking of tags,3) undesired immune responses to tags, and4) the strong influence of scattering light and an autofluorescence background, which allow the assessment of only superficial microvessels with a slow flow velocity. Our contribution in this field includes the development of PA and PT flow cytometry (PAFC/PTFC) for in vivo real-time detection of individual circulating cells either with intrinsic absorbing markers (e.g., Hb in RBCs or melanin in melanoma cells) or synthetic gold and other nanoparticles as PT and PA contrast agents (7–10, 19–26). PT spectroscopy has demonstrated capabilities to identify several Hb chemical subtypes (e.g., sulpha-, met-, CO- or HbS [in sickle cells]) (27). Our group also developed Raman and integrated PA-PT-Raman spectroscopy and imaging for in vivo identification with chemical specificity of individual cells and nanoparticles (28, 29). However, the application of PT and PA techniques to the study of blood rheology has not yet been reported. Here, we analyze the capabilities of a combined platform (Fig. 1), for integrating high-speed PT, PA, and optical imaging for in vivo real-time monitoring of multiple rheological parameters at the single-blood-cell level.
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Here, we describe a platform integrating PT and PA techniques with conventional optical imaging for real-time, studies of blood rheology. This platform, verified in an animal model, represents a further development of our previous findings in which we applied PT/PA techniques to the in vivo investigation of lymph rheology, permitting the assessment of the “lymphocrit,” cell deformability in lymph flow, and lymph viscosity (22, 37). The diagnostic principles are based on the sensitivity of PT/PA signal parameters (e.g., amplitude, temporal profile, and dynamic behavior) to the local concentration and spatial distribution of Hb in RBCs and RBCs in flow, that is related to dynamic changes in the hemorheological properties of blood flow.
This technology can provide dynamic multiparameter monitoring and imaging of RBC aggregation, alterations of RBC deformability, and Ht. We also demonstrated in vivo high-speed (up to 10,000 fps) and high-resolution (up to 300 nm) imaging for the continuous monitoring of transient RBC deformability in fast blood flow. The relationship between cellular functions and mechanical properties suggests that cell deformability can be used as a marker of early or latent stages of different pathological processes. We demonstrated that the integration of PAFC, PTFC and PT imaging provides in vivo the real-time rheological status of circulating blood in microvessels through dynamic measurements of multiple parameters, including RBC aggregation, Ht, wall shear rate, dynamic shape of moving RBCs, RBC deformability, and intracellular distribution of Hb. We also found for the first time that this technology could, noninvasively and without labeling, assess relatively large vessels and detect changes in two important rheological parameters— Ht and RBC aggregation— together. Finally the capability of the PT/PA technical platform was tested ex vivo and in vivo in a mouse model of human sickle cell disease. We believe that in vivo PA blood testing and rapid (510 min) PT image cytometry (38, 40) of RBCs ex vivo using a small drop of blood can add valuable information about diagnosis of sickle disease.
Although we demonstrated proof of this concept using the visible-spectral range, where the absorption of Hb is maximal, our previous finding suggests that individual RBCs can produce detectable PA signals in near-infrared (NIR) range, in which light achieves maximum penetration into biotissues (20,21). The further development of PT/PA imaging by increasing its speed and resolution could provide accurate measurement of blood vessel diameter for calculation of the Ht.
In addition to measuring the main determinants of hemorheology that we demonstrated here, the capabilities of PTFC and PAFC can be significantly extended to measure many more rheological parameters such as blood viscosity as a complex parameter that depends on Ht, RBC deformability, RBC aggregation, and concentrations of plasma proteins with high molecular weights. PT/PA monitoring of plasma proteins can be performed in far NIR range, where they provide readable absorption in the absence of a Hb background (e.g., in capillaries).
Previously, we and others demonstrated that PT and PA techniques can measure blood flow velocity (9, 19, 25, 51), which could be the basis for calculating the velocity profile and shear rate of the vascular wall. Such spatially resolved measurements of wall shear rates have the potential to answer long-standing questions about ways that blood shear forces exerted on endothelial cells contribute to physiological and pathological processes, such as atherogenesis and angiogenesis (44). For example, it is well known that endothelial cells' sensing of the shear stress gradient promotes activation of genes responsible to measure, vasoregulation, and proliferation. Previously, there were no methods to measure the shear stress gradient in vivo. Because we found that PT and PA signals from a Hb solution and RBCs under the same conditions are significantly different, PAFC and PTFC can likely be used to diagnose pathological intravascular hemolysis in vivo.
A novel diagnostic platform using safe laser parameters and a label-free, noninvasive approach can be quickly translated to use in humans. Indeed, compared with other optical modalities, noninvasive PA methods offer higher resolution, sensitivity, and penetration depth (up to 3 cm), and the minimally invasive delivery of laser radiation through tiny fibers (9) could allow the assessment of potentially any site in the human body. The clinical prototype could be in the form of a portable fiber-based device (e.g., see prototype in the supplementary information for Ref. 9) that is placed over different vessels ranging from capillaries in the nail or eye to large vessels in the hand or neck area. Patient management may be improved though ultrasensitive point-of-care monitoring of blood rheology in vivo, instead of existing invasive time-consuming in vitro testing (see Introduction).
The testing that we have described could be useful for the early, sensitive diagnosis of a broad spectrum of diseases accompanied by alterations of multiple rheological parameters in the acute stage of critically ill patients, in cardiovascular dysfunction, cancer, sickle cell disease, infections, and intoxications. Potential applications include use in emergency departments and intensive care units for patients with shock, trauma, anemia, renal insufficiency, neurological deficit, or congestive heart failure.
This testing would also enable physicians to individualize therapy and assess the therapeutic efficacy of existing and novel treatments. The diagnostic approach may integrate new PT/PA rheological testing with techniques that we recently developed including 1) PT/PA Raman flow cytometry (28, 29), which provides additional diagnostic parameters of blood chemistry through differences in PA Raman vibrational contrast from blood components; 2) identification of spectral signatures of the internal chemical structures of different forms of Hb (e.g., sulpha-, met-, CO-, or HbS [in sickle cells]) in the NIR region in the background of reduced and oxygenated Hb (27); 3) high-speed spectral imaging for measurement of oxygenation and Hb at the single-RBC level compared to bulk integrated parameters; 4) PA detection of circulating clots; and 5) monitoring of changes in the volume of circulating blood. Hypothetically, many standard blood parameters that are currently measured by conventional in vitro blood testing can be measured in real-time in vivo more quickly (a few minutes vs. several hours) and with greater sensitivity.