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.
Scanning Integrated PT/PA Microscope-Cytometer
The integrated setup (Fig. 1A) was built on the technical platform of an inverted microscope (IX81, Olympus America., USA), with an incorporated PT, PA, fluorescent and transmission modules, and a tunable optical parametric oscillator (OPO, Opolette HR 355 LD, OPOTEK, Carlsbad, CA) with the following parameters: spectral range of 410–2,500 nm, pulse width, 5 ns; pulse energy, up to 2 mJ; pulse energy stability, 3–5%; pulse repetition rate, 100 Hz; line width ∼0.5 nm (30, 31). Pump laser energy was controlled by a powermeter (PE10-SH, OPHIR, Israel). In addition to the tunable OPO, the setup was equipped with a high pulse repetition rate laser (Model: LUCE 532, Bright Solutions, Italy) with the parameter set: wavelength, 532 nm; maximum pulse energy, 100 μJ; pulse width, 5 ns; and pulse repetition rate, up to 50 kHz. Laser-induced PA waves were detected with an unfocused ultrasound transducer (model 6528101, 3.5 MHz, 5.5 mm in diameter; Imasonic, Besançon, France) or a focused cylindrical transducer (model V316-SM, 20 MHz, focal length, 12.5 mm; Panametrics-NDT, Olympus) and amplifier (5662B, 5 MHz, gain 60 dB and 5678, 40 MHz, gain 60 dB, respectively; both from Panametrics- NDT, Olympus). Warm water or ultrasound gel was applied for better acoustic matching between the transducer and the samples. The PA signal under single laser pulse had a bipolar shape that was transformed into a pulse train due to reflection and resonance effects.
In PT thermolens schematic, laser-induced temperature-dependent variations of the refractive index around absorbing zones caused defocusing of a continuous-wave collinear He-Ne laser (model 117A, Spectra-Physics; wavelength, 633 nm; 1.4 mW) probe beam, leading to a reduction in the beam's intensity at its center, detected by a photodiode with preamplifier (PDA36A, 40 dB amplification, ThorLabs). The linear PT thermolens signal under single laser pulse demonstrated the standard fast-rising unipolar peak associated with rapid (picosecond-nanosecond scale) sample heating and a slower (microsecond scale) tail corresponding to sample cooling. PT/PA mapping of the sample was realized by scanning the sample with two-dimensional (X-Y) translation stage (H117 ProScan II, Prior Scientific) with a positioning accuracy of 50 nm. Scanning along the Z-axis was performed by moving the microscopic objective axially. The lateral resolution was ∼0.7μm with a 20× objective, and a ∼300 nm with 100× oil immersion objective.
The microscope was equipped with a standard fluorescence module (Olympus). A high resolution, cooled, color CCD camera (DP72, Olympus) was used for the navigation of laser beams and for the verification of PT data with fluorescent images using specific fluorescent tags. High-resolution (300 nm) transmission digital microscopy (TDM), fitted with a high-speed (up to 40,000 fps) CMOS (MV-D1024-160-CL8; Photonfocus AG, Lachen, Switzerland) and high sensitive CCD (Cascade: 512; Photometrics, Roper Scientific) cameras were used for label-free imaging blood vessels with circulating and adhered clots. Conventional absorption spectra were obtained by fiber spectrophotometer (USB4000, Ocean Optics, USA).
The acquisition and processing of PT and PA data using the OPO system with relatively low pulse rate (100 Hz) was done by digitizer (PCI-5124, 200 MSPS, 12-bit, National Instruments, Austin, TX) installed in a workstation (Precision 690, Dell, Round Rock, TX). This system was used to acquire signals from the photodetector, transducer and energy meter. Synchronization of the OPO, signal acquisition, processing and control of translation stage was implemented in a single software module (customized software based on LabView 8.5, National Instruments, USA). The signals were recorded also with a Tektronix TDS 3032B oscilloscope.
For both, PT and PA, signal acquisitions using high pulse repetition rate lasers (10 kHz), we installed a digitizer (AD484, 120 MSPS, 14-bit, 4DSP Inc, Reno, NV) onto the workstation (Precision T7500, Dell, Round Rock, TX). The custom FPGA (Field Programmable Gate Array) firmware on the digitizer calculated the spectrum of the signals after each trigger, and transferred a certain region of interest (ROI) in these spectrums for further processing. A MATLAB-based (Mathworks, Natick, MA) program was developed to analyze the recorded data. It was used to determine the baseline, the threshold level, the peaks above threshold, and to provide statistical analysis. A continuous real-time monitoring of the acquired signals was displayed in the time increment of 100 ms, which enabled the convenient presentation of each increment as a full-screen frame. The appearance of a distinct sharp, negative peak indicated the passage of a clot (Fig. 1D). A high pass filtering was applied because the sharp, negative PA peak consisted mainly of high frequency components in the range of 100 Hz – 5 MHz. Artifacts secondary to movement of blood vessel (e.g., caused by beating heart or breathing) generated low frequency noise below 100 Hz. The relatively smooth baseline after filtering represented PA background due to absorption by red blood cells (RBCs) (Fig. 1D). Each trace was analyzed for the presence of peaks with height exceeding a defined threshold. The stable baseline associated with the background absorption by blood was accurately subtracted allowing observation of the small negative peaks (Fig. 2) at a different number of averaged PA signals (Fig. 3).
Preparation of Clots In Vitro
White clots were produced by platelet aggregation using collagen applied to a 0.5-mL aliquot of platelet-rich plasma or stabilized blood. Red clots as a result of hyperaggregation of RBCs in PBS or whole blood samples were created by Dextran500. Blood samples were collected from human donors (Institutional Review Board-approved protocol at the University of Arkansas for Medical Sciences) and animals (protocol approved by the University of Arkansas for Medical Sciences Institutional Animal Care and Use Committee).
Platelet-rich plasma was prepared via centrifugation of whole blood at 200g for 6 min to remove RBCs. Platelets were washed via centrifugation of platelet-rich plasma at 1,000g for 10 min at room temperature. The pellet was resuspended in PBS to the desired concentration. Single RBCs were prepared by their isolation through initial centrifugation of whole blood after the removal of plasma and leukocyte layers.
Blood circulation was modeled in vitro by using a flow module with the flow speed of 0–14 cm/s (KD Scientific, Holliston, MA) in glass tubes filled with 1 mL of human or mouse blood. For calibration of negative contrast, blood samples were spiked with transparent polymer beads with diameters ranged from 12 to 200 μm.
In vivo experiments involved a noninvasive mouse ear model (nude mice nu/nu), mouse carotid artery model, mouse myocardial infarction model (occlusion of left coronary artery in the anesthetized wild-type mice three weeks earlier) and rat mesentery model (Sprague-Dawley rats), in accordance with protocols approved by the University of Arkansas for Medical Sciences Institutional Animal Care and Use Committee. We selected rat mesentery as an ideal minimally invasive animal model because it has a thin tissue layer which allows clear identification of single-blood vessels that can be assessed with high-resolution optical (transmission) and PT techniques. After anesthesia (ketamine/xylazine, 50/10 mg/kg, i.m.), the animal was laparotomized and mesentery was exteriorized on a customized, heated (37.7°C) microscope stage. It was then suffused with warmed Ringer's solution (37.7°C, pH 7.4) containing 1% bovine serum albumin. Three to five small arteries and veins with different diameters were examined using PA and PT techniques over a period of 120 min.
Experiments with mice were performed on the thin (250 μm) mouse ear with well-distinguished blood vessels at 50–100 μm depth and 50–150 μm diameter. The anesthetized animal was placed on the heated microscope stage with a topical application of water on the ear for acoustic matching of the ultrasound transducer and the tissue. The carotid artery was examined through the incision in skin.
The clots were produced by an intravenous (i.v.) injection of collagen (No.385 Collagen, Chrono-Log; 100 μg/kg), Dextran500 and by a topical application of strip with FeCl3 on large vessels (e.g., carotid artery) for 2–3 min. Fluorescent labeling of the platelets was obtained by i.v. injection of carboxyfluorescein diacetate succinimidyl ester (CFSE; 20 μg per a mouse; excitation 489 nm, emission 520 nm). Fluorescent video monitoring of red clots was obtained after labeling plasma with fluorescein isothiocyanate (FITC) - dextran.
A minimum of three animals were used for each experiment unless otherwise noted. Results are expressed as a mean ± standard error. Spearman correlations for which P < 0.05 were considered statistically significant. MATLAB 7.0.1 (MathWorks, Natick, MA), and LabVIEW (National Instruments) were used for the statistical calculations. Data were summarized as the mean, standard deviation (SD), median, inter-quartile range, and full range. Comparisons of PA and PT data were done via scatter plot in conjunction with Spearman correlation analysis.
Principle of Label-Free PA Detection of Circulating Clots In Vivo
Laser irradiation of blood vessels (Fig. 1A) creates a constant PA background (Fig. 1D) determined by high-absorbing hemoglobin in RBCs. The background level depends on laser energy, absorption spectra (Fig. 1C) (32) and hemoglobin concentration which in turn depends on number of RBCs (i.e., hematocrit) in the detected (irradiated) volume. To be detected using the conventional positive contrast mode, the target must have higher absorption compared with blood background. When this target (e.g., RBC-rich red clot) passes through the irradiated volume blood, a transient increase in local absorption results in a sharp positive PA peak (Fig. 1D, left). Inversely, in the negative contrast mode, the target must have lower absorption than the whole blood as in the case of platelets (33). When a low-absorbing, platelet-rich, RBC-depleted clot passes the laser beam, a transient decrease in local absorption is accompanied by formation of a narrow PA negative dip as the marker of a white clot (Fig. 1D, middle). A mixed clot with both RBC-rich (i.e., high-absorbing) and platelet-rich (i.e., low-absorbing) local zones should produce a pattern of positive and negative PT/PA signals (Fig 1D, right). Indeed, according to our calculations, dense aggregate of 5 RBCs has a volume of ∼450 fL (corpuscular volume of single human RBC is 90 fL) with total hemoglobin amount of 150 pg (∼30 pg/cell), while the same volume of whole blood at the typical hematocrit of 40% contains just 2 RBCs with 60 pg of hemoglobin (32). Thus, the expected positive contrast PA signal from dense RBC-rich (or hemoglobin from damaged RBCs) clot is approximately 2.5 times higher as compared with background signal from randomly distributed RBCs in the same volume. In contrast, the platelet-rich clot zone should dramatically decrease signals from blood background to almost zero level (i.e., negative contrast).
Optimization of PA and PT Parameters Using Animal and Human Blood Samples
This phenomenological model was verified by developing a high speed PAFC using a high pulse rate laser and fast PA signal acquisition system. We selected a spectral range of 530–600 nm with a strong blood absorption band (Fig. 1C) (23, 32) that provides relatively large PA signals from blood background, allowing monitoring of negative contrast in a wide dynamic range. Use of a PA and PT scanning cytometry (Figs. 4 and 5) (30, 31) revealed that PA/PT signals from a single human and animal platelet were approximately three orders of magnitude less than the signals from a single RBC at the same laser energy (Fig. 5A, first and second columns). Compared with a single platelet, PA/PT signals from platelet-rich clot were 10- to 20-fold higher (Figs. 4 and 5A, second and third columns); nevertheless, the difference between signals from the white clots and individual RBCs was still significant, around 50- to 100-fold. In contrast, RBC aggregates produced PA/PT signals with amplitudes which were significantly higher than from single RBCs, and correlated with the aggregate sizes (Fig. 5A, first and forth columns). As a result, laser scanning of whole human blood with white clots demonstrated distinct negative PA/PT contrasts (Fig. 5B), and positive contrasts when laser beam scanned red clot (Fig. 5C). The mixed (i.e., red and white) clots were recognized by the specific pattern of positive and negative (i.e., combined) PA/PT contrasts associated with RBC- and platelet-rich areas (Fig. 5D). Using low absorbing beads of different known sizes as a white clot phantom in vitro in static and flow conditions, we determined that PA signal amplitudes are dependent on bead-size with readable signal levels down to 12-μm beads (Figs. 5E and 6). These results were confirmed using blood samples in a 120-μm-thick slide with real clots of different sizes determined independently by optical imaging (Figs. 4 and 5F).
In Vivo Flow Cytometry of Circulating White Clots
To provide a proof-of-concept in vivo, we first selected a rat mesentery model (34) with distinguishable single-blood vessels which are assessable simultaneously with transmission and PT/PA techniques (Figs. 7 and 8A–C). Intravenous injection of collagen led to the formation of circulating platelet-rich clots (8), which were detected by a decrease in both, PT and PA signals (Fig. 8B), and verified by high-speed optical imaging. The PA negative dip level, its width and signal rate varied within a large range (20–100%, 10–100 ms, and 0.1–1.0 clot/min, respectively), providing real-time clot enumeration and indicating a wide heterogeneity in clot size and velocity. Starting 10–15 min after injection, some clots adhered to the vessel wall (Figs. 7 and 8A). Thrombi were frequently localized in bifurcation zones. They slightly oscillated under blood flow pressure leading, in particular, to the chaotic motion of the intra-thrombotic platelets. In the most cases, the thrombi showed stable adhesion to the endothelium accompanied by their dynamic composition modification. Initially, the adherent clots represented almost transparent structures with no evidence of RBCs incorporated into the clot. Over time, some leukocytes (white blood cells [WBCs]) and RBCs were directed from flow to the thrombus by stretching or rotating motions and adhered to the thrombus. This eventually led to an increase in thrombus size and promoted the transformation from white thrombus to red one (Fig. 7). This finding indicates that the clots initially circulate as platelet-rich white clots with minimal interaction with RBCs which move with similar velocity as platelets and minimal collisions with platelets. However, as circulating RBCs collide with the adherent clot, RBCs start adhering to the platelet-fibrin rich mesh. The dynamic transformation of white clot into a mixed (white and red) clot was monitored by high speed optical imaging, and PA and PT cytometry as a switching of purely negative signal to a pattern of positive and negative contrast signals. These results are in agreement with in vitro data (Figs. 5B and 5D).
In Vivo Detection of Red Clots
Intravenous injection of Dextran500 as a well-established inductor of circulating RBC aggregates led to the expected appearance of transient positive PA contrast signals (Fig. 8C, bottom, right). The mean amplitude of these signals was two times larger than the background signals from intact blood. The presence of moving RBC aggregates was confirmed also by fluorescent imaging after intravenous injection of FITC-dextran: aggregates as large dark spots were clearly distinguished on the background of bright fluorescent plasma (i.e., negative fluorescent contrast; Fig. 8C, top right) and differed from nonaggregated blood (Fig. 8C, top left).
Noninvasive High-Speed PA Flow Cytometry of Clots
Noninvasive PA monitoring of circulating clots was performed using mouse ear model. The formation of moving collagen-induced white clots was verified with fluorescent imaging by targeting of platelets in vivo with CFSE fluorescent dye (Fig. 8D). High-speed PAFC with a high pulse repetition rate laser showed negative PA dips (Fig. 8E) suggesting clot formation at the rate of 1-2 clots/min and negative contrast levels ranging from 12 to 59%. The thrombi later embolized in remote organs, such as lungs (Fig. 9) of these animals, in agreement with published data (8).
PA Detection of Clots in a Carotid Artery
To verify the capability of PAFC to detect clots in the mouse carotid artery (Fig. 8F), we used a well-established model of thrombus (15) by application of FeCl3 directly to the exposed artery (n = 3; Fig. 8F). We observed a gradual decrease of PA signals in the carotid artery over 2–5 min, suggesting clot formation, accompanied by changes in the shape and the width of PA signals (Fig. 8G, right) compared to control (Fig. 8G, left).
Diagnosis of Circulating Clots in Preclinical Model of Myocardial Infarction
A real-time dynamics of circulating clots were monitored in mice with myocardial infarction created by total ligation of left coronary artery. The readable transient PA signals were observed with different patterns of negative, positive, and combined contrasts (Fig. 8H) compared to no signals in control normal mice. According to previously described findings, the origin of these signals is likely related to the formation of white, red, and mixed clots, respectively.
Prevention of False Positivity Related to White Blood Cells
In order to estimate the influence of WBCs on a PT/PA signals among RBCs, blood samples were placed in the wells with a varied thickness from 5 to 120 μm. The presence of leukocyte in the detected volume was controlled by transmission imaging. As expected, the scanning of a focused laser beam across the sample provided readable PT/PA negative contrasts in signal-traces when the leukocytes were more than 30% of slide thickness. In vivo PT monitoring of blood capillaries and microvessels showed that leukocytes or plasma in capillaries between distinguished single RBCs provided a detectable negative contrasts in the vessels with a mean diameter of 10–25 μm only. These in vivo results were in agreement with in vitro results indicating that the influence of single WBCs in the detected area on negative contrast is minimal in vessels with a mean diameter of 30–40 μm and more.
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.
The authors thank Evgeny Shashkov and Scott Fergusson for their help in experiments.