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. At known the irradiated volume and clot size which could be measured independently with imaging, the signal width provides information of clot velocity. When compared to the previously observed negative PA effects and contrasts, mostly in non-biologic samples (17
), the dynamic negative PT/PA contrast modes introduced here may have broad biological and clinical applications.
In comparison to adhered clots (4
) 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 the 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). In particular, it has been estimated that 795,000 individuals suffer stroke per year in the US (36
). Most strokes occur unexpectedly are either thromboembolic (approx. 80%) or hemorrhagic (20%). The approximately 30% of stroke causes (238,500 people for each year) are not clear identified. The common cardiac causes of thrombo-embolism (approx. 80% of thromboembolic strokes) are atrial fibrillation, or left ventricular clots formed in patients with myocardial infarction and cardiomyopathic disorders (37
). In some cases circulating clots are considered as fragments shedding from disrupted atherosclerotic plaques (39
). Nevertheless, the most clots in the carotid arteries and during acute myocardial infarction are platelet-rich, also called 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 contracting or paradoxically contracting (reference normal myocardium). In these conditions, the clot formed is often rich in fibrin, RBCs with some incorporation of platelets and leukocytes. Although the size of the emboli that cause strokes and other systemic thrombo-embolic disorders varies, the most thrombi are approximately 100 μm in diameter. From these clinically established facts many patients who are prone to develop thromboembolic phenomenon may have micro-thrombi in circulation. As such, demonstrated here techniques can be potentially applied to select patients with hyper-production of circulating clots, to provide real-time multiparameter (e.g., composition, sizes, velocity, ability to adhesion) monitoring of these clots and to find correlation the presence of circulating clots in systemic circulation with the incidence of clinically identifiable stroke, heart attack or/andother thrombo-embolic disorders.
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 (, right). In addition, the clot velocity and size at a fixed known beam diameter can be estimated through PA negative peak width (23
) and PA negative dip level (verified by optical imaging) respectively. In particular, for the selected blood vessels the clot velocity it was ranged from 100 μm/s to 3 mm/s, while blood velocity estimated by high speed imaging (34
) was around 2–3 mm/s. These data indicate the possible presence of rolling clots which velocity is much slower compared to blood flow (14
Although maximum negative contrast was observed at wavelength of 532 nm, a 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 ().
Toward clinical translation portable watch-like flow cytometer with negative contrast mode and a built-in a small diode laser and transducer (23
) could be developed for assessment of circulating clots in different blood vessels from capillaries in the hand, lip or eye area to large coronary artery in the neck area vessels. 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
). 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, a PA signal is less sensitive to the position of the laser beam on the skin and to the 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 pre-cursor 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 realtime 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 abbreviation 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.