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Conventional photothermal (PT) and photoacousic (PA) imaging, spectroscopy, and cytometry are preferentially based on positive PT/PA effects, when signals are above background. Here, we introduce PT/PA technique based on detection of negative signals below background. Among various new applications, we propose label-free in vivo flow cytometry of circulating clots. No method has been developed for the early detection of clots of different compositions as a source of thromboembolism including ischemia at strokes and myocardial infarction. When a low-absorbing, platelet-rich clot passes a laser-irradiated vessel volume, a transient decrease in local absorption results in an ultrasharp negative PA hole in blood background. Using this phenomenon alone or in combination with positive contrasts, we demonstrated identification of white, red, and mixed clots on a mouse model of myocardial infarction and human blood. The concentration and size of clots were measured with threshold down to few clots in the entire circulation with size as low as 20 μm. This multiparameter diagnostic platform using portable personal high-speed flow cytometer with negative dynamic contrast mode has potential to real-time defining risk factors for cardiovascular diseases, and for prognosis and prevention of stroke or use clot count as a marker of therapy efficacy. Possibility for label-free detection of platelets, leukocytes, tumor cells or targeting themby negative PA probes (e.g., nonabsorbing beads or bubbles) is also highlighted. © 2011 International Society for Advancement of Cytometry
Thromboembolism is a known medical problem and detection of circulating clots of different composition is likely to help in defining risk for thromboembolic complications (1–8). However, existing diagnosis of circulating clots is far from ideal. Ex vivo methods of detecting blood clots are cumbersome, invasive, time-consuming, limited by discrete time-points, and are insensitive because of the use of small volume blood samples (6–8). Measurement of platelet aggregation, for example, provides indirect evidence of in vivo clot formation only. The procedure requires anticoagulants, such as ethylene diamine tetraacetic acid (EDTA) or sodium citrate, for blood collection that may lead to artifacts, and this method ignores the influence of platelet-activating factors elicited by the vessel wall (6, 7). These problems could be solved by noninvasive assessments of larger blood volumes in vivo; however, existing diagnostic techniques, such as magnetic resonance imaging (MRI) and positron emission tomography (PET) can only detect fixed or slowly moving large (macro) clots and do not allow diagnosis of fast moving small micro-clots (9–16). Fluorescent imaging can identify rolling clots in some experimental models (16); however, translation of this method to humans is problematic because of the toxicity of fluorescent tags, influence of autofluorescence background, and assessment of superficial microvessels only with slow flow velocity. Pulse Doppler ultrasound is a promising technique for detection of circulating clots, but this technique cannot assess clot composition, detect clots smaller than 50–100 μm, and avoid errors due to artifacts (11, 12).
As an alternative, the photothermal (PT) and photoacoustic (PA) methods (also termed optoacoustic [OA]) based on detection of laser-induced heat and accompanied thermo-elastic acoustic waves provide greater sensitivity and spatial resolution for detection and imaging of nonfluorescent cells in vitro and in deep (up to 3 cm) vessels in vivo compared with the other optical modalities. Currently, PA technique is one of the fastest growing areas of biomedical imaging which have been effective for diagnosis of malignancy, visualization of blood vessels, and detection of circulating tumor cells and infections (17–23). The clinical significance and safety of PA devices has been demonstrated in humans (18), including monitoring of blood oxygenation in large (1–1.5 cm) human blood vessels with depth of 1.5–2.5 cm (24). PA imaging has been shown to detect adhered thrombi in a study of phantoms and veins in vitro (25, 26). However, PA detection of circulating clots in vivo has not yet been reported due to the lack of fast PA signal acquisition algorithms. As a result, schematic and methodology for detection of low-absorbing fast moving clots within a strong absorption background of blood have not been yet available.
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In this article, we introduce a new label-free, noninvasive real-time PA method, which has the potential to identify composition, size and rate of circulating clots by analyzing PA signal shape, width, and rate, respectively. For known irradiated volume and clot size which could be measured independently with imaging, the signal width provides information on clot velocity. When compared to the previously observed negative PA effects and contrasts, mostly in nonbiologic samples (17, 27, 28), the dynamic negative PT/PA contrast modes introduced here may have broad biological and clinical applications.
In comparison to adherent clots (4, 14, 15, 25, 35), the behavior of circulating clots and their clinical role are still poorly understood. For example, some circulating clots remain undetectable (fast moving micro-clots) unless they result in a clinical phenomenon, and their significance for human disease remains unclear. It is not known as to what sizes of circulating clots correlate with thromembolism and are functionally relevant. We hope that our diagnostic platform can help to answer on some of these and other important questions.
In particular, it is not currently clear that detection of circulating clots may be an indicator of a risk for stroke and myocardial infarction. Nevertheless, there is no doubt that the blood samples of most patients with myocardial infarction and unstable angina have high concentration of active pro-coagulants and increased number of aggregated platelets. Circulating clots are a well-established cause of venous and pulmonary thromboembolism, ischemic stroke, and transient ischemic attacks (e.g., in individuals with atrial fibrillation). It is estimated that 795,000 individuals suffer a stroke every year in the US (36). Most strokes are ischemic (87%); the remaining, hemorrhagic strokes constitute 10 to 15% of all strokes. A large percentage (≈30%) of ischemic strokes are embolic, with examples of sources of emboli being atrial fibrillation, or left ventricular clots developing in patients with acute myocardial infarction and other cardiomyopathic and hypercoagulable disorders (37,38). In some cases circulating clots are considered as fragments shedding from disrupted atherosclerotic plaques on which platelet aggregates had formed (39,40). This is the case of most clots in the carotid arteries and in acute myocardial infarction (white clots). At times, these clots, formed in the chronically “sick” heart, are a result of turbulence in the left atrium and ventricle as the tissues are either poorly, or paradoxically contracting (reference normal myocardium). In these circumstances, clots formed are often rich in fibrin, RBCs, platelets and leukocytes. Although the size of clots that cause strokes and other systemic thrombo-embolic disorders varies, most clots are approximately 50-200 μm in diameter (5). Many patients who are prone to develop thrombo-embolic phenomenon may have micro-thrombi in the circulation. As such, the techniques demonstrated here could be potentially applied to select patients with hyper-production of circulating clots, provide real-time multiparametric (e.g., composition, sizes, velocity, adhesion ability) monitoring of these clots, and finally attempt to correlate the presence of these clots in the systemic circulation with the incidence of stroke, heart attack, and other thrombo-embolic phenomena.
Our study revealed the capability of time-resolved PA and PT technique to identify white, red, and mixed clots through signals with negative, positive, and combined contrasts respectively. In addition, the clot velocity and size at a fixed known beam diameter can be estimated through PA negative peak width (23, 41) and PA negative dip level respectively. In particular, for the selected blood vessels the clot velocity ranged from 100 μm/s to 3 mm/s, while blood velocity estimated by high speed imaging (34, 42) was around 2–3 mm/s. These data indicated the possible presence of rolling clots which velocity is much slower compared with blood flow (14).
Although maximum negative contrast was observed at wavelength of 532 nm, high sensitivity of PA technique allows in the future to use the near-infrared range with better penetration of light into tissue where absorption contrast between RBCs and platelet is still significant (Fig. 1C).
Toward clinical translation, portable watch-like flow cytometer with negative contrast mode and a built-in small diode laser and transducer (23) could be developed for assessment of circulating clots in different blood vessels from microvessels in the hand, lip or eye area to large carotid artery in the neck area. Indeed, the capability of PA technique to assess deep and large human blood vessels at depths of 1–3 cm and 0.2–1 cm in diameter in vivo is already well-documented (18, 24). We also have verified the capability of our prototype to assess mouse aorta with diameter of 0.7–0.9 mm using a high frequency focused cylindrical ultrasound transducer (23).
The main challenge in clinical application is to choose the correct laser geometry to achieve overlapping of an entire diameter of a vessel for detection of all clots moving through a vascular cross-section. We found that at the orientation of a linear beam shape across the vessel compared to along the vessel or circular beam shape, PA signals are less sensitive to the position of the laser beam on the skin and to natural human movements (e.g., due to breathing and overall motion). Thus, lateral resolution is determined in superficial tissue by optical focal parameters (5–10 μm) and in deeper tissue by ultrasonic focal parameters (20–80 μm, at frequency 10–75 MHz). As a result, the cylindrical focal configuration allows us to keep a minimal detected volume (due to high lateral resolution) and simultaneously assess the whole cross section of a vessel.
In conclusion, after further validation of described technology, clot detection may be potentially used: (1) as a prognostic marker or a precursor for a thrombo-embolic events such as myocardial infarction and stroke; (2) to study the dynamics of platelet aggregation directly in the bloodstream in pathologic states such as infections and cancer; (3) for real-time assessment of therapeutic efficacy of pharmacologic compounds by quantifying clots before, during and after therapy; and (4) to study clots as well as tumor cells, bacteria, viruses, or microparticles in circulation by targeting them with PA negative (i.e., nonabsorbing) probes such as functionalized beads, nano- and microbubbles, or liposomes. This technique may improve detection limit of clots or microparticles as small as 20 μm in circulating blood. Applications where this technology is more likely to be applied during cardiopulmonary bypass, problems that occur during hemodialysis, complications of thrombolytic therapy of occluded AV dialysis shunts, or evaluating complications of intracardiac right to left shunting. We hope that it can be used also for prognosis of stroke risk, and if successful, for its prevention by well-time therapy.