Detection Of Microbubbles Research Articles

Cardiovascular Molecular Imaging with Contrast Ultrasound: Principles and Applications

Methods for imaging the molecular or cellular profile of tissue are being developed for all forms of non-invasive cardiovascular imaging. It is thought that these technologies will potentially improve patient outcomes by allowing diagnosis of disease at an early-stage, monitoring disease progression, providing important information on patient risk, and for tailoring therapy to the molecular basis of disease. Molecular imaging is also already assuming an important role in science by providing a better understanding of the molecular basis of cardiovascular pathology, for assessing response to new therapies, and for rapidly optimizing new or established therapies. Ultrasound-based molecular imaging is one of these new approaches. Contrast-enhanced ultrasound molecular imaging relies on the detection of novel site-targeted microbubbles (MB) or other acoustically active particles which are administered by intravenous injection, circulate throughout the vascular compartment, and are then retained and imaged within regions of disease by ligand-directed binding. The technique is thought to be advantageous in practical terms of cost, time, and ease of use. The aim of this review is to discuss the molecular participants of cardiovascular disease that have been targeted for ultrasound imaging, general features of site-targeted MB, imaging protocols, and potential roles of ultrasound molecular imaging in cardiovascular research and clinical medicine.

Toward in vivo detection of hydrogen peroxide with ultrasound molecular imaging

We present a new class of ultrasound molecular imaging agents that extend upon the design of micromotors that are designed to move through fluids by catalyzing hydrogen peroxide (H2O2) and propelling forward by escaping oxygen microbubbles. Micromotor converters require 62 mm of H2O2 to move – 1000-fold higher than is expected in vivo. Here, we aim to prove that ultrasound can detect the expelled microbubbles, to determine the minimum H2O2 concentration needed for microbubble detection, explore alternate designs to detect the H2O2 produced by activated neutrophils and perform preliminary in vivo testing. Oxygen microbubbles were detected by ultrasound at 2.5 mm H2O2. Best results were achieved with a 400–500 nm spherical design with alternating surface coatings of catalase and PSS over a silica core. The lowest detection limit of 10–100 μm was achieved when assays were done in plasma. Using this design, we detected the H2O2 produced by freshly isolated PMA-activated neutrophils allowing their distinction from naïve neutrophils. Finally, we were also able to show that direct injection of these nanospheres into an abscess in vivo enhanced ultrasound signal only when they contained catalase, and only when injected into an abscess, likely because of the elevated levels of H2O2 produced by inflammatory mediators.

An optical system for detecting 3D high-speed oscillation of a single ultrasound microbubble

As contrast agents, microbubbles have been playing significant roles in ultrasound imaging. Investigation of microbubble oscillation is crucial for microbubble characterization and detection. Unfortunately, 3-dimensional (3D) observation of microbubble oscillation is challenging and costly because of the bubble size-a few microns in diameter-and the high-speed dynamics under MHz ultrasound pressure waves. In this study, a cost-efficient optical confocal microscopic system combined with a gated and intensified charge-coupled device (ICCD) camera were developed to detect 3D microbubble oscillation. The capability of imaging microbubble high-speed oscillation with much lower costs than with an ultra-fast framing or streak camera system was demonstrated. In addition, microbubble oscillations along both lateral (x and y) and axial (z) directions were demonstrated. Accordingly, this system is an excellent alternative for 3D investigation of microbubble high-speed oscillation, especially when budgets are limited.

Quantitative analysis of carotid plaque vasa vasorum by CEUS and correlation with histology after endarterectomy

Intraplaque neovascularization and vasa vasorum (VV) proliferation contribute in the progression and rupture of atherosclerotic lesions. Contrast enhanced ultrasonography (CEUS) has been reported to attain data regarding intraplaque neovessels and VV. However, whether the detection of microbubbles by CEUS within atherosclerotic plaques truly represents microvessels is a point of concern. We aimed to evaluate stable and unstable carotid artery plaque (CAP) VV pattern by CEUS and its correlation with histology and immunochemistry. Patients with CAP scheduled for plaque endarterectomy were enrolled. CAP was initially identified by conventional ultrasonography and subsequently CEUS (harmonic ultrasound imaging with simultaneous intravenous contrast agent injection) was performed. The recorded image loops were evaluated by a semi-automated method. Plaque specimens were excised and underwent histological and immunochemical (for CD34, Vascular Endothelial Growth Factor, CD68 and CD3 antibodies) analysis. Fourteen patients (67.6 ± 10.2 years, 10 males) with a 86.9 ± 11.5 % degree of carotid artery stenosis were evaluated. Histology showed that half of the plaques were unstable. Enhancement of plaque brightness on CEUS was significant for both stable and unstable plaque subgroups (p = 0.018 for both). Immunochemistry showed that microvessels, as assessed by CD34 antibody, were more dense in unstable vs. stable plaques (36.6 ± 17.4 vs. 13.0 ± 7.2 respectively, p = 0.002). However, correlation between plaque brigthness enhancement on CEUS and microvessel density was significant only for stable (r = 0.800, p = 0.031) plaques. The identification of brightness enhacement during CEUS in carotid atherosclerotic plaques may not always reflect the presence of VV.

The use of twinkling artifact of Doppler imaging to monitor cavitation in tissue during high intensity focused ultrasound therapy

In high intensity focused ultrasound (HIFU) therapy, it is important to monitor the presence and activity of microbubbles in tissue during treatment. The current methods—passive cavitation detection (PCD) and B-mode imaging—have limited sensitivity, especially to small-size, non-violently collapsing microbubbles. Here, a new method for microbubble detection is proposed, based on “twinkling” artifact (TA) of Doppler imaging. TA occurs when color Doppler ultrasound is used to image hard objects in tissue (e.g., kidney stones) and is displayed as brightly colored spots. As demonstrated recently, TA can be explained by irregular scattering of the Doppler ensemble pulses from the fluctuating microbubbles trapped in crevices of the kidney stone. In this work, TA was used to detect cavitation in tissue and in polyacrylamide gel phantoms during pulsed 1 MHz HIFU exposures with different peak negative pressures (1.5–11 MPa). At each pressure level, the probability of cavitation occurrence was characterized using TA and the broadband signals recorded by PCD, aligned confocally with the HIFU transducer. The results indicate that TA is more sensitive to the onset of cavitation than conventional PCD detection, and allows for accurate spatial localization of the bubbles. [Work supported by RFBR and NIH (EB007643, 1K01EB015745, and R01CA154451).]

The effect of boundary proximity on the fundamental and subharmonic emissions from individual microbubbles at higher frequencies

It is recognized that the proximity of a boundary can influence the dynamic behavior of acoustically stimulated microbubbles. In a biomedical ultrasound context, this is relevant to molecular imaging with targeted microbubbles, and when microbubbles are near vessel walls or contained within microvessels. Theoretical models have recently been developed to examine these effects, but experimental work has been more limited and primarily focused on the assessment of resonant frequency effects rather than its impact on nonlinear behavior, which is perhaps more relevant to imaging applications. Understanding this behavior is important to improving microbubble detection and for the quantitative interpretation of contrast images. With the use of an optical trap, this study experimentally investigates the effect of boundary proximity (0 to 200 µm) and boundary stiffness (Opticell and agarose) on fundamental and subharmonic emissions from individual Definity and MicroMarker bubbles at 11 MHz. The scattered pressure dependence on proximity from an Opticell boundary resulted in an oscillatory dependence, while from an agarose boundary resulted in a decreasing fundamental and an increasing subharmonic response with increasing distance from the boundary. These experimental findings are not entirely captured by basic analytical simulations, likely suggesting that more complex numerical models may be required.

Contrast enhancement of medical ultrasound imaging with reversal phase-inversion pulse technology

A new ultrasound contrast imaging technique was proposed for eliminating the harmonic components from the emission signal transmitted by the broadband ultrasonic system. Reversal phase-inversion pulse was used for the first time to separate the contrast harmonics from the harmonics in the emission signal to improve the detection of contrast micro-bubbles. Based on the nonlinear acoustic theory of finite-amplitude effects and the associated distortion of the propagating wave, the Bessel-Fubini series model was applied to describe the nonlinear propagation effects of the reversal phase-inversion pulse, and the Church’s equation for zero-thickness encapsulation model was used to produce the scattering-pulse of the bubble. For harmonic imaging, the experiment was performed using a 64-element linear array, which was simulated by Field II. The results show that the harmonic components from the emission signal can be completely cancelled, and the harmonics generated by the nonlinear propagation of the wave through the tissue, can be reduced by 15–30 dB. Compared with the short pulse, the reversal phase-inversion pulse can improve the contrast and definition of the harmonic image significantly.

Detection of Micro-Bubbles in Thin Polymer Films by Means of Acoustic Resonant Spectroscopy

This paper describes an acoustical method for detecting small scattering sources in thin polymer films with high sensitivity. The technique monitors the acoustic resonance occurring between water, the film and a metallic plate, and an accurate measurement of the ultrasonic attenuation in the film at the resonant frequency is made. Micro-bubbles in the film cause the ultrasonic attenuation in the film to increase.

The use of twinkling artifact of Doppler imaging to monitor cavitation in tissue during high intensity focused ultrasound therapy.

In high intensity focused ultrasound (HIFU) therapy, it is important to monitor the presence and activity of microbubbles in tissue during treatment. The current methods, - passive cavitation detection (PCD) and B-mode imaging - have limited sensitivity, especially to small-size, non-violently-collapsing microbubbles. Here, a new method for microbubble detection is proposed, based on "twinkling" artifact (TA) of Doppler imaging. TA occurs when Color Doppler ultrasound is used to image hard objects in tissue (e.g., kidney stones), and is displayed as brightly colored spots. As demonstrated recently, TA can be explained by irregular scattering of the Doppler ensemble pulses from the fluctuating microbubbles trapped in crevices of the kidney stone. In this work, TA was used to detect cavitation in tissue and in polyacrylamide gel phantoms during pulsed 1 MHz HIFU exposures with different peak negative pressures (1.5-11 MPa). At each pressure level, the probability of cavitation occurrence was characterized using TA and the broadband signals recorded by PCD, aligned confocally with the HIFU transducer. The results indicate that TA is more sensitive to the onset of cavitation than conventional PCD detection, and allows for accurate spatial localization of the bubbles. Work supported by RFBR and NIH (EB007643, 1K01EB015745, R01CA154451).

Intravascular ultrasound detection and delivery of molecularly targeted microbubbles for gene delivery

We are investigating the combination of microbubble-based targeted drug delivery and intravascular ultrasound (IVUS) imaging as a potential therapy to reduce incidence of restenosis following stent placement in atherosclerotic coronary arteries. The goal of these studies was to determine whether IVUS could be used to detect targeted microbubbles and enhance drug/gene delivery through targeting. Quiescent vascular smooth muscle cells (SMCs) were stimulated with cytokine IL-1β to induce the inflammatory cell surface marker vascular cell adhesion molecule 1 (VCAM-1). Molecular-targeted (VCAM-1 Ab or IgG control Ab), fluorescent-labeled microbubbles were conjugated with plasmid DNA expressing green fluorescent protein (GFP, pMax-GFP) and exposed to the inflamed SMCs under flow to measure adhesion compared with control microbubbles. Gene delivery was performed using a modified IVUS catheter to generate 1.5-MHz ultrasound at 200 kPa. Detection of adherent microbubbles to inflamed SMCs in culture and flow chambers was measured using an IVUS catheter and scanner. VCAM-1-targeted microbubbles enhanced adhesion to inflamed SMCs 100-fold over nontargeted microbubbles. Compared with noninflamed SMCs, VCAM-1-targeted microbubbles exhibited a 7.9-fold increase in adhesion to IL-1β-treated cells. Targeted microbubbles resulted in a 5.5-fold increase in plasmid DNA transfection over nontargeted microbubbles in conjunction with a focused 2.54-cm (1-in) diameter 1-MHz transducer and also enhanced transfection by the modified IVUS transducer at 1.5 MHz. Targeted microbubbles (at a density of 3 × 10⁴ microbubbles/mm²) increased IVUS image intensity 13.2 dB over non-microbubble-coated surfaces. Rupture of microbubbles from the modified IVUS transducer resulted in a 53% reduction in image intensity. Taken together, these results indicate that IVUS may be used to detect targeted microbubbles to inflamed vasculature and subsequently deliver a gene/drug locally.

Ultrasound Microbubbles for Molecular Diagnosis, Therapy, and Theranostics

Ultrasound imaging is clinically established for routine screening examinations of breast, abdomen, neck, and other soft tissues, as well as for therapy monitoring. Microbubbles as vascular contrast agents improve the detection and characterization of cancerous lesions, inflammatory processes, and cardiovascular pathologies. Taking advantage of the excellent sensitivity and specificity of ultrasound for microbubble detection, molecular imaging can be realized by binding antibodies, peptides, and other targeting moieties to microbubble surfaces. Molecular microbubbles directed against various targets such as vascular endothelial growth factor receptor-2, vascular cell adhesion molecule 1, intercellular adhesion molecule 1, selectins, and integrins were developed and were shown in preclinical studies to be able to selectively bind to tumor blood vessels and atherosclerotic plaques. Currently, the first microbubble formulations targeted to angiogenic vessels in prostate cancers are being evaluated clinically. However, microbubbles can be used for more than diagnosis: disintegrating microbubbles emit acoustic forces that are strong enough to induce thrombolysis, and they can also be used for facilitating drug and gene delivery across biologic barriers. This review on the use of microbubbles for ultrasound-based molecular imaging, therapy, and theranostics addresses innovative concepts and identifies areas in which clinical translation is foreseeable in the near future.

A preliminary in vitro assessment of polymer-shelled microbubbles in contrast-enhanced ultrasound imaging

This paper focuses on the use of poly (vinyl alcohol)-shelled microbubbles as a contrast agent in ultrasound medical imaging. The objective was an in vitro assessment of the different working conditions and signal processing methods for the visual detection (especially in small vessels) of such microbubbles, while avoiding their destruction. Polymer-shelled microbubbles have recently been proposed as ultrasound contrast agents with some important advantages. The major drawback is a shell that is less elastic than that of the traditional lipidic microbubbles. Weaker echoes are expected, and their detection at low concentrations may be critical. In vitro experiments were performed with a commercial ultrasound scanner equipped with a dedicated acquisition board. A concentration of 100 bubbles/mm 3, excitation pressure amplitudes from 120 kPa to 320 kPa, and a central frequency of 3 MHz or 4.5 MHz were used. Three multi-pulse techniques (i.e., pulse inversion, contrast pulse sequence based on three transmitted signals, and contrast pulse sequence in combination with the chirp pulse) were compared. The results confirmed that these microbubbles produce a weaker ultrasound response than lipidic bubbles with a reduced second-order nonlinear component. Nevertheless, these microbubbles can be detected by the contrast pulse sequence technique, especially when the chirp pulse is adopted. The best value of the contrast-to-tissue ratio was obtained at an excitation pressure amplitude of 230 kPa: although this pressure amplitude is higher than what is typically used for lipidic microbubbles, it does not cause the rupture of the polymeric contrast agent.

Microbubble detection following hyperbaric chamber dives using dual-frequency ultrasound

Venous gas emboli (VGE) can be readily detected in the bloodstream using existing ultrasound methods. No method currently exists to detect decompression-induced microbubbles in tissue. We hypothesized that dual-frequency ultrasound (DFU) could detect these microbubbles. With DFU, microbubbles are driven with two frequencies: a lower "pump" (set to the resonant frequency of the desired bubble size) and a higher "image" frequency. A bubble of the resonant size emits the sum and difference of the two transmitted frequencies. For this study we used a pump frequency of 2.25 MHz and an image frequency of 5.0 MHz, which detects bubbles of roughly 1-10 μm in diameter in a water tank. Four anesthetized swine were pressurized at 4.5 ATA for 2 h and decompressed over 5 min, inducing moderate to very severe VGE scores. Four sites on the thigh of each swine were monitored with DFU before and after the dives. A single mock dive was also performed. The number of sites returning signals consistent with microbubbles increased dramatically after the chamber dive (P < 0.01), but did not change with the mock dive. The increase in DFU signal after the chamber dive was sustained and present at multiple sites in multiple swine. This research shows for the first time that decompression-induced tissue microbubbles can be detected using DFU and that DFU could be used to monitor decompression-induced microbubbles at multiple sites on the body. Additionally, DFU could be used to track the time course of microbubble formation and growth during decompression stress.

Microbubbles in imaging: applications beyond ultrasound

Since their introduction as ultrasound contrast agents, microbubbles have demonstrated the potential to revolutionise the use of ultrasound at the bedside. Aside from clinical application, where microbubbles are used to enhance ultrasonic assessment of myocardial perfusion, they have demonstrated potential in an exciting host of pre-clinical ultrasound imaging and therapeutic applications. These include the ability to target specific cellular markers of disease, provide dynamic blood flow estimation, deliver localised chemotherapy, potentiate the mechanisms of gene therapy, enhance lesion ablation through cavitation, and spatiotemporally permeabilise the blood-brain barrier. The unique and flexible construction of microbubbles not only enables a variety of ultrasound applications, but also opens the door to detection of microbubbles with modalities other than ultrasound. In this review, non-ultrasound imaging applications utilizing microbubbles are discussed, including MRI, PET, and DEI. These various imaging approaches illustrate novel applications of microbubbles, and may provide the groundwork for future multi-modality imaging or image-guided therapeutics.

Ultrasound Molecular Imaging of Cardiovascular Disease

Molecular imaging with ultrasound contrast agents relies on the detection of microbubbles within diseased tissue. Microbubbles produce an acoustic signal owing to their resonant properties in an ultrasound field. Microbubble targeting is accomplished by either manipulating the microbubble shell for attachment of microbubbles to activated leukocytes, or by conjugation of disease-specific ligands to the microbubble surface. Inflammation, angiogenesis, and thrombus formation are central pathophysiologic processes in many cardiovascular diseases and produce phenotypic changes in the vascular compartment that can be imaged with targeted ultrasound contrast agents. In the future, targeted contrast ultrasound could aid in the diagnosis of atherosclerosis, myocardial ischemia, transplant rejection, and thrombosis syndromes and could be used for assessing angiogenesis.

“Blue” photonics: optics in the sea

The oceans and coastal zones of the world are vitally important resources for many aspects of human life. Their exploitation as sources of food dates back to the beginnings of civilisation, but in relatively more recent times, by comparison, our oceans have become valuable sources of energy. The oil and gas industries are well known examples of how the oceans can be exploited to provide us with our fossil fuel needs. More recently, wind, tidal and wave power are coming to the fore as we look more towards renewable sources of energy. We should also not forget the extraction of other mineral resources. However, the gathering and farming of food is still the most significant treasure that the oceans hold for us; and its control, management and sustainability are vital aspects of sea utilisation. There are many significant challenges which still face us in our drive to understand and utilise this unique environment and to exploit the natural resources contained within it. New techniques and instruments are continually being developed to help us explore and enhance our appreciation of the sea and how we depend upon it. Foremost amongst these is the increased use of optical techniques or photonics that help to us to extract information about the nature of the oceans, its constituents and its behaviour. In this special issue of the Journal of the European Optical Society we focus on what we term “Blue” Photonics: in other words, the use and understanding of optics in the sea (and also in rivers and lakes). Such a title embraces a wide range of aspects of optics and light in the oceans from basic optical propagation through water to the design of complex instruments which enable us to measure its properties and behaviour. The chosen papers highlight major topics in photonics applied to marine science and engineering from fundamental science through marine optics, optical metrology, biological optics, environmental optics to technology and instrumentation. A number of papers are related to the measurement of ocean water properties like the transparency measurements using the Secchi Disc (Wernand) or the colour evaluation with the Forel-Ule comparator (Wernand) and its change as a function of time (Wernand) caused by pollution or the presence of micro-organism. Another two papers deal with the development of new measurement methods and their influencing parameters. An interesting method in this area is the measurement of reflectance scattering with the application to the measurement of waves on the ocean surface (Creanor et al) as well as to the determination of solutes in the water. A complementary method is the measurement of light fluctuations in different depths under the surface giving an excellent approach to the wave evaluation (Hieronymi). The detection of micro-bubbles in the subsea environment is discussed by Zielinski et al. A new measurement method too is the Laser Line Scan method in the CW mode as well as in pulsed mode (Caimi and Dalgleish). This method increases the resolution and is robust against environmental influences and scans a larger volume. Imaging methods are another area of underwater metrology systems. Systems like the LOKI system (Schulz et al) are used to image Plankton. The papers here act only as a primer for the vast amount of work now being undertaken in Blue Photonics and we hope that they will serve as a catalyst for increased use of optics in the sea.

Design of chirp excitation waveform for dual-frequency harmonic contrast detection

Tissue background suppression is essential for harmonic detection of ultrasonic contrast microbubbles. To reduce the tissue harmonic amplitude for improvement of contrast-to-tissue ratio (CTR), the method of third harmonic (3f(0)) transmit phasing uses an additional 3f(0) transmit signal to provide mutual cancellation between the frequency-sum component and the frequency-difference component of tissue harmonic signal. Chirp excitation can further improve the SNR in harmonic imaging without requiring an excessive transmit pressure and thus reduce potential bubble destruction. However, for effective suppression of tissue harmonic background in 3f(0) transmit phasing, the 3f(0) chirp waveform has to be carefully designed for the generation of spectrally matched cancellation pairs over the entire second harmonic band. In this study, we proposed a chirp waveform suitable for the method of 3f(0) transmit phasing, the different-bandwidth chirp signal (DBCS). With the DBCS waveform, the frequency-difference component of tissue harmonic signal becomes a chirp signal similar to its frequency-sum counterpart. Thus, the combination of the DBCS waveform with the 3f(0) transmit phasing can markedly suppress the tissue harmonic amplitude for CTR improvement together with effective SNR increase of contrast harmonic signal. Our results indicate that, as compared with the conventional Gaussian pulse, the DBCS waveform can provide 6-dB improvement of SNR in 3f(0) transmit phasing with a CTR increase of 3 dB. Nevertheless, the limitation of available transmit bandwidth and the frequency-dependent attenuation can degrade the performance of the DBCS waveform in tissue suppression. The design of the DBCS waveform is also applicable to other dual-frequency imaging techniques that rely on the harmonic generation at the difference frequency.

A Method for Differentiating Targeted Microbubbles in Real Time Using Subharmonic Micro-Ultrasound and Interframe Filtering

This study introduces a new method for differentiating targeted microbubbles in the presence of flowing microbubbles and tissue using micro-ultrasound. The method relies on subharmonic (SH) imaging for segmenting microbubble signals from tissue signals, and low-pass interframe filtering for segmenting bound targeted microbubbles from flowing microbubbles. The method is evaluated with 30 frames per second SH B-mode imaging in vitro, using a wall-less vessel flow phantom. The SH B-mode cineloops were postprocessed using an interframe moving average filter to segment the regions of bound microbubbles on the inner surface of the vessel phantom. The bound bubbles were then disrupted with sufficiently high ultrasound pressures, so that the dynamic process of targeted microbubble binding under flowing conditions could be observed. These preliminary results show that the proposed method is a feasible solution to the challenge of differentiating targeted microbubbles in the presence of tissue and freely flowing microbubbles at high frequencies, which in turn should improve the specificity of targeted microbubble detection. (E-mail: aneedles@visualsonics.com)

0078: Evaluation of Microvascular Physiology

Contrast-enhanced ultrasound is based on the detection of acoustically-active microbubbles. Intravital microscopy has demonstrated that these contrast agents are confined to the vascular space and that their rheologic profile is similar to that of erythrocytes. Hence, these agents are ideal tracers for evaluating microvascular perfusion. The ability to parametrically non-invasively evaluate blood flux rate through tissue and absolute microvascular blood volume has also proved to be a valuable asset for evaluating complex microvascular adaptations in health and disease.

Discussion of the application of finite Volterra series for the modeling of the oscillation behavior of ultrasound contrast agents

Ultrasound contrast agents consist of microbubbles with diameters in the micrometer range. Excited by ultrasound, these bubbles exhibit highly nonlinear oscillation. While well developed physical models for microbubble oscillation exist, the efficiency of pulse sequences for sensitive microbubble detection is discussed based on simple mathematical models of general nonlinearity. Typically, Taylor series are used to model microbubble nonlinearity for the development of detection schemes. Recently, pulse sequences were proposed which exploit nonlinear memory of microbubbles, a property that cannot be modeled by a Taylor series but can be explained using a Volterra series. Therefore, this paper discusses and evaluates the usage of Volterra series for the modeling of the scattering behavior of contrast agent microbubbles. A numerically stable linear estimation algorithm is implemented to determine a third order Volterra model for a free gas bubble with a resting radius r 0 = 1 μ m . For insonification pressures up to 100 kPa, the identified model allowed for a mean-square error of less than −16 dB with respect to the reference signal. Analysis of the response to narrowband signals showed that the achievable mean-square error is further reduced for the bandwidth available to typical ultrasound transducers used for clinical diagnostics.