Color Doppler Ultrasound Twinkling Artifact Research Articles

Ultrasound diagnosis and treatment of heterotopic ossification

Heterotopic ossification (HO), or the presence of bone in soft tissues, can occur after musculoskeletal trauma, causing pain and reduced mobility. However, even the most sensitive diagnostic modality requires 2–3 weeks after initiation to detect HO, and the only treatment is surgical resection after HO matures (>2 years). Here, we evaluate the color Doppler ultrasound twinkling artifact for early diagnosis of HO and focused ultrasound (fUS) for treatment of early HO. We began by evaluating twinkling and fUS parameters in ossified cell culture. We then evaluated imaging and treatment parameters in mice with HO. Results show that twinkling correlated well with the presence of ossified cells; although the exact size/number of ossified cells required for twinkling was unclear. In mice, twinkling was found to detect HO in some mice as early as 3 days-post-injection, which is earlier than other imaging modalities. For treatment, fUS at 1.07-MHz and 0.2% duty-cycle with p + /p− = 17/7 MPa was found to be most successful at disrupting mineralizations with little damage to surrounding cells. Mice treated with fUS were found to have less advanced HO compared to sham mice. These results highlight the promise of ultrasound for the diagnosis and treatment of early HO. [Work supported by CDMRP PR201164].

The effect of gas composition on the color Doppler ultrasound twinkling artifact

The twinkling artifact, or twinkling, appears when imaging kidney stones and other mineralizations with Doppler ultrasound and is caused by scattering off crevice microbubbles. Previous work found that twinkling on in vivo kidney stones increased or decreased with elevated oxygen or carbon dioxide, respectively. However, it is unclear whether these results are from biological adaptions to the changing respiratory gas and thus if in vitro mineralizations are similarly affected. Here, cholesterol, calcium phosphate, and uric acid crystals were grown in vitro and imaged in deionized water using a Vantage-128 research ultrasound system with a Philips/ATL L7-4 transducer. Five of each crystal composition were imaged for one minute at different dissolved gas concentrations. By bubbling gas through water, dissolved oxygen ranged from 2 to 20 mg/L while dissolved carbon dioxide ranged from 30 to 290 mg/L. Twinkling on cholesterol crystals increased by 70% and 140% when oxygen and carbon dioxide levels were maximized, respectively. In contrast, twinkling on calcium phosphate and uric acid was not significantly affected by either gas. These results suggest that changes in twinkling from increased gas concentrations are dependent on crystal composition and are likely affected by biological adaptations in vivo. [Work supported by NSF-CAREER-1943937 and NSF-GRFP-DGE1255832.]

The effect of crystal composition and environment on the color Doppler ultrasound twinkling artifact

Objective. Pathological mineralizations form throughout the body and can be difficult to detect using conventional imaging methods. Color Doppler ultrasound twinkling highlights ∼60% of kidney stones with a rapid color shift and is theorized to arise from crevice microbubbles as twinkling disappears on kidney stones at elevated pressures and scratched acrylic balls in ethanol. Twinkling also sometimes appears on other pathological mineralizations; however, it is unclear whether the etiology of twinkling is the same as for kidney stones. Approach. In this study, five cholesterol, calcium phosphate, and uric acid crystals were grown in vitro and imaged in Doppler mode with a research ultrasound system and L7-4 transducer in water. To evaluate the influence of pressure on twinkling, the same crystals were imaged in a high-pressure chamber. Then, the effect of surface tension on twinkling was evaluated by imaging crystals in different concentrations of surfactant (1%, 2%, 3%, 4%) and ethanol (10%, 30%, 50%, 70%), artificial urine, bovine blood, and a tissue-mimicking phantom. Main results. Results showed that all crystals twinkled in water, with cholesterol twinkling significantly more than calcium phosphate and uric acid. When the ambient pressure was increased, twinkling disappeared for all tested crystals when pressures reached 7 MPa (absolute) and reappeared when returned to ambient pressure (0.1 MPa). Similarly, twinkling across all crystals decreased with surface tension when imaged in the surfactant and ethanol (statistically significant when surface tension <22 mN m−1) and decreased in blood (surface tension = 52.7 mN m−1) but was unaffected by artificial urine (similar surface tension to water). In the tissue-mimicking phantom, twinkling increased for cholesterol and calcium phosphate crystals with no change observed in uric acid crystals. Significance. Overall, these results support the theory that bubbles are present on crystals and cause twinkling, which could be leveraged to improve twinkling for the detection of other pathological mineralizations.

The effect of surface tension on the color Doppler ultrasound twinkling artifact

Imaging crystalline structures with Doppler ultrasound can produce a rapid color shift, termed the twinkling artifact, that can assist in diagnosing pathological mineralizations such as kidney stones, heterotopic ossification, gout, and breast microcalcifications. Twinkling is theorized to arise from scattering off surface crevice microbubbles, which are affected by the surface tension between the bubble and surrounding medium. In this study, we evaluated the effect of surface tension on twinkling in pure crystals. Cholesterol, calcium phosphate, and uric acid crystals were grown in vitro (n = 5 each) and imaged with a Philips/ATL L7-4 transducer and Vantage-128 research ultrasound system. Crystals were imaged in water while varying surfactant concentration (0%–4%) leading to surface tensions that ranged from 45–72 mN/m. Surface tension of the solution was determined by measuring the contact angle of 0.1-mL droplet on an acrylic sheet exposed to air. As the concentration of surfactant increased, twinkling was found to decrease by ∼20% for cholesterol and calcium phosphate and ∼10% for uric acid. These results continue to support the crevice bubble theory of twinkling and suggest the importance of surface tension when evaluating minerals with the twinkling artifact. [Work supported by NSF-CAREER-1943937 and NSF-GRFP-DGE1255832.]

Investigating the color Doppler twinkling artifact as an early detector of cellular mineralization

Heterotopic ossification (HO) occurs when bone develops in areas where bone does not usually exist, often appearing after blast injuries or other musculoskeletal trauma. Current methods of diagnosis, such as three-phase bone scintigraphy, x-ray, or CT scans, take several weeks after the trauma before HO is visible. The color Doppler ultrasound twinkling artifact has been shown to detect the presence of kidney stones and other crystals. Twinkling could provide a more sensitive method of HO detection, which would allow for earlier diagnosis and treatment and limit HO severity. In our research, human bone marrow-derived stem cells (HBMSCs) were plated onto collagen scaffolds and cultured in osteogenic media to promote mineralization orgrowth media (controls). The cells were imaged every 3rd day from day 10 – day 25 using Phillips L7-4, L12-5, and Vermon L22-14v transducers and a Vantage-128 research ultrasound system; I/Q data were bulk saved for quantitative analysis. Samples with mineralization on 0.5% of their surface showed a 17% increase in Doppler magnitude compared to controls, suggesting that the twinkling artifact is a sensitive method for detection of mineralization in vitro. Further studies will investigate whether this method is effective in a murine model. [Work supported by DoD CDMRP PR201164.]

Color Doppler imaging of pure crystals in hyperbaric conditions

The color Doppler ultrasound twinkling artifact is a rapid color shift that appears on some kidney stones and other pathological mineralizations during ultrasound imaging. Twinkling on kidney stones is primarily attributed to scattering from stabilized microbubbles as increasing hydrostatic pressures has been shown to decrease twinkling. However, it is unknown whether microbubbles are also present and cause twinkling on other forms of pathological mineralizations or pure crystals. In this study, 5 cholesterol and 5 calcium phosphate crystals were grown in vitro from supersaturated solutions and imaged in a custom-built, hydrostatic pressure chamber with a Philips/ATL L7-4 transducer and Vantage-128 research ultrasound system. Hydrostatic pressure was increased to a maximum of 14 MPa while saving IQ data at 1 fps for evaluation of Doppler power. On all 10 imaged crystals, twinkling was found to decrease by approximately 90% when pressures reached 4 MPa absolute. Twinkling reappeared when the pressure was returned to atmospheric levels. These results suggest that bubbles are present on pure crystals and give rise to the twinkling artifact. [Work supported by NSF CAREER 1943937 and NSF GRFP DGE1255832.]

Evaluation of Stone Features That Cause the Color Doppler Ultrasound Twinkling Artifact

The color Doppler ultrasound twinkling artifact is a rapid color shift that appears on 43%–96% of kidney stones. Surface microbubbles on kidney stones are theorized to cause twinkling as exposure to elevated static pressures of 0.41–1.13 MPa (approximately 0.5–1 times diagnostic ultrasound pressure and 5–10 times ambient pressure) reduced twinkling. However, it is unclear what external and internal stone features support bubbles. Thirteen ex vivo kidney stones were scanned with color Doppler ultrasound at 2.5, 5 and 18.5 MHz. Select stones were imaged with environmental scanning electron microscopy or underwater micro-computed tomography to evaluate features that may cause twinkling. Results revealed that the lower frequencies produced larger volumes of twinkling. Condensation first occurred in the smallest (∼1 µm diameter) surface pores and may be indicative of where bubbles form. Gas pockets were seen inside two of three tested stones that may contribute to twinkling. Overall, these results provide evidence of cavity structures both externally and internally and their correlation to the twinkling artifact. This indicates that microbubbles may be present on and within kidney stones and may contribute to the twinkling artifact.

Evidence of Microbubbles on Kidney Stones in Humans

The color Doppler ultrasound twinkling artifact has been found to improve detection of kidney stones with ultrasound; however, it appears on only ∼60% of stones. Evidence from ex vivo kidney stones suggests twinkling arises from microbubbles stabilized in crevices on the stone surface. Yet it is unknown whether these bubbles are present on stones in humans. Here, we used a research ultrasound system to quantify twinkling in humans with kidney stones in a hyperbaric chamber. Eight human patients with non-obstructive kidney stones previously observed to twinkle were exposed to a maximum pressure of 4 atmospheres absolute (ATA) while breathing air, except during the 10-min pause at 1.6 ATA and while the pressure decreased to 1 ATA, during which patients breathed oxygen to minimize the risk of decompression sickness. A paired one-way t-test was used to compare the mean twinkle power at each pressure pause with baseline twinkling, with p < 0.05 considered to indicate significance. Results revealed that exposure to 3 and 4 ATA of pressure significantly reduced twinkle power by averages of 35% and 39%, respectively, in 7 patients (p = 0.04); data from the eighth patient were excluded because of corruption. This study supports the theory that microbubbles are present on kidney stones in humans.

MP10-12 THE COLOR DOPPLER ULTRASOUND TWINKLING ARTIFACT AND MICROBUBBLES ON KIDNEY STONES IN HUMANS: A PILOT STUDY

INTRODUCTION AND OBJECTIVE:The color Doppler ultrasound twinkling artifact, which highlights kidney stones with rapidly changing color, has been shown to increase the sensitivity and specificity of...

Color Doppler ultrasound twinkling in pathological biomineralizations

The color Doppler ultrasound twinkling artifact has been observed on some kidney stones as well as various sites in the body where pathological biomineralization can occur. On kidney stones, twinkling was recently attributed to stabilized crevice microbubbles, which suggests that microbubbles may also be present on other minerals. However, it is unknown how crystal structure, composition, and the surrounding environment influences the presence and location of microbubbles and twinkling. Here, crystals found in pathological biomineralization (i.e., uric acid, cholesterol, calcium phosphate, and calcium oxalate), were grown in vitro, embedded in a tissue-mimicking phantom, and imaged with a research ultrasound system at 5 and 18.5 MHz. Doppler power was quantified and compared between crystal composition (n = 5 per type). We found twinkling was strongest in cholesterol crystals and weakest in calcium phosphate crystals. Micro-computed tomography (µCT) of an ex vivo calcium oxalate kidney stone (i.e., a heterogeneous crystal) submerged in water revealed the presence of gas within internal microcracks, which was confirmed by reducing the hydrostatic pressure in a subsequent µCT scan. Future work includes exposing pure in vitro crystals to µCT and ultrasound with changes in hydrostatic pressure to evaluate for internal gas pockets that may contribute to twinkling.

Microbubbles on kidney stones contribute to the twinkling artifact in humans

The color Doppler ultrasound twinkling artifact, a rapid color change that highlights kidney stones, was recently attributed to surface crevice microbubbles on ex vivo stones because twinkling was affected by changes in hydrostatic pressure. However, it was unclear whether crevice bubbles existed on in situ human kidney stones, and, if so, how smooth-surfaced stones could harbor the crevice bubbles that give rise to twinkling. Here, 8 human subjects with known kidney stones were exposed to 4 atmospheres absolute (ATA) while breathing air inside a hyperbaric chamber; twinkling was monitored and quantified with a research ultrasound system. At 3 and 4 ATA, twinkling was significantly reduced by an average of 35% and 39%, respectively (p = 0.04). Then, ex vivo kidney stones that twinkled were exposed to micro-computed tomography (μCT) and hypobaric pressures while submerged in water. Regions of low x-ray attenuation within the stone (i.e., microcracks) at 1 ATA expanded when the pressure was reduced to 0.1 ATA. These results support the theory that microbubbles are present on kidney stones in the human body and that microbubbles may be internal as well as external to the kidney stone. [Work supported by NSBRI through NASA NCC 9-58 and NIH DK043881.]

The effect of crystal chemical composition on the color Doppler ultrasound twinkling artifact

The color Doppler ultrasound twinkling artifact, or localized areas of rapid color shifts, has been used to aid in the diagnosis of kidney stones. Gallstones and some atherosclerotic plaques have also been shown to twinkle, suggesting that crystals, regardless of chemical composition, may harbor the stable microbubbles that are hypothesized to cause twinkling. Here, uric acid, cholesterol, and calcium phosphate crystals, which are often present in sites of pathological biomineralization, were grown in the lab using previously developed techniques and imaged with the Verasonics® research ultrasound system using Philips/ATL P4-2 and Verasonics L22-14 transducers. The magnitude of the Doppler signals was quantified and compared amongst the three crystal chemical compositions. Preliminary results show that all three crystal types displayed twinkling with the strongest magnitude appearing in the cholesterol crystals. These results suggest that twinkling is very sensitive in the presence of crystals regardless of the chemical composition. Future work will explore the use of histotripsy to mechanically fractionate crystals as a novel treatment for pathological biomineralization.The color Doppler ultrasound twinkling artifact, or localized areas of rapid color shifts, has been used to aid in the diagnosis of kidney stones. Gallstones and some atherosclerotic plaques have also been shown to twinkle, suggesting that crystals, regardless of chemical composition, may harbor the stable microbubbles that are hypothesized to cause twinkling. Here, uric acid, cholesterol, and calcium phosphate crystals, which are often present in sites of pathological biomineralization, were grown in the lab using previously developed techniques and imaged with the Verasonics® research ultrasound system using Philips/ATL P4-2 and Verasonics L22-14 transducers. The magnitude of the Doppler signals was quantified and compared amongst the three crystal chemical compositions. Preliminary results show that all three crystal types displayed twinkling with the strongest magnitude appearing in the cholesterol crystals. These results suggest that twinkling is very sensitive in the presence of crystals regardless ...

Characterizing tissue calcifications using the color Doppler ultrasound twinkling artifact

Rapid Doppler shifts highlight some kidney stones with a rapid change of color in ultrasound imaging in what is termed the “twinkling artifact.” While many hypotheses exist to describe the origin of twinkling, the currently accepted hypothesis is that surface microbubbles are stabilized in the crevices on the surface of the kidney stone. The objective here is to evaluate the distribution of bubbles stabilized on the kidney stone surface and to determine whether other calcified tissues display the twinkling artifact. A Verasonics® research ultrasound system with the Verasonics® L22-14 and Philips/ATL L7-4 and P4-2 transducers was used to quantify twinkling on kidney stones and in minerals deposited by osteogenic stem cells over a range of frequencies. Preliminary results on kidney stones suggest that surface roughness and chemical composition influence the magnitude of twinkling. Osteogenic stem cells were also found to twinkle when minerals were present; correlating twinkling magnitude with the quantity and distribution of mineral deposition is a topic of ongoing research. The appearance of twinkling on even small tissue calcifications indicates ultrasound is very sensitive to mineral deposition and suggests that bubbles may be byproduct of the calcification process.

The effect of hypobaric pressure on the kidney stone twinkling artifact

Recently, our group proposed the color Doppler ultrasound twinkling artifact originates from stable crevice bubbles on the kidney stone surface because overpressure suppressed twinkling on ex vivo stones (Lu et al., Ultrasound Med. Biol. 2013). However, the hypothesis is not fully accepted because the bubbles were not directly observed. Here, the submicron-sized bubbles predicted by the crevice-bubble hypothesis are enlarged in ex vivo kidney stones by exposure to a pre-focal, off-axis lithotripter pulse (p + = 1.5 MPa, p-=2.5 MPa) or hypobaric static pressures (0.021 MPa, absolute) to simultaneously capture their appearance by high-speed photography and ultrasound imaging. In rough stones that twinkle, consecutive lithotripter pulses caused more than 50% of bubbles to grow reproducibly from specific locations on the stone surface, suggesting the bubbles were pre-existing. Conversely, on smooth stones that did not twinkle, repeated lithotripter pulses initiated bubbles from varying locations on the stone surface. Similarly, upon exposure to hypobaric static pressures, the simple expectation that twinkling would increase by enlarging bubbles largely held for rough-surfaced stones but was inadequate for smoother stones. These results suggest a correlation between kidney stone surface topography or stable surface crevice bubbles and twinkling. [Work supported by NSBRI through NASA NCC 9-58 and NIH DK043881.]

The role of trapped bubbles in kidney stone detection with the color Doppler ultrasound twinkling artifact

The color Doppler ultrasound twinkling artifact, which highlights kidney stones with rapidly changing color, has the potential to improve stone detection; however, its inconsistent appearance has limited its clinical utility. Recently, it was proposed stable crevice bubbles on the kidney stone surface cause twinkling; however, the hypothesis is not fully accepted because the bubbles have not been directly observed. In this paper, the micron or submicron-sized bubbles predicted by the crevice bubble hypothesis are enlarged in kidney stones of five primary compositions by exposure to acoustic rarefaction pulses or hypobaric static pressures in order to simultaneously capture their appearance by high-speed photography and ultrasound imaging. On filming stones that twinkle, consecutive rarefaction pulses from a lithotripter caused some bubbles to reproducibly grow from specific locations on the stone surface, suggesting the presence of pre-existing crevice bubbles. Hyperbaric and hypobaric static pressures were found to modify the twinkling artifact; however, the simple expectation that hyperbaric exposures reduce and hypobaric pressures increase twinkling by shrinking and enlarging bubbles, respectively, largely held for rough-surfaced stones but was inadequate for smoother stones. Twinkling was found to increase or decrease in response to elevated static pressure on smooth stones, perhaps because of the compression of internal voids. These results support the crevice bubble hypothesis of twinkling and suggest the kidney stone crevices that give rise to the twinkling phenomenon may be internal as well as external.

Etiology of the color Doppler ultrasound twinkling artifact on in situ human kidney stones

Hyperbaric pressures of 0.3–10 MPa (absolute) have been shown to reduce the magnitude of the color Doppler ultrasound twinkling artifact on ex vivo human kidney stones, supporting the hypothesis that surface crevice microbubbles cause twinkling. For the first time, we investigate the etiology of twinkling on in situ human kidney stones. Eight human subjects with kidney stones known to twinkle were imaged with a Philips/ATL P4-2 transducer and Verasonics ultrasound system for 45 minutes while inside a hyperbaric chamber. Subjects breathed ambient air while the pressure in the chamber cycled to a maximum pressure of 0.4 MPa, with a scheduled decompression stop at 0.16 MPa where subjects breathed pure oxygen. Preliminary results show no change in the twinkling amplitude at 0.4 MPa compared to the baseline measurements before pressurization; however, a statistically significant increase in the twinkling amplitude was observed when subjects breathed pure oxygen at 0.16 MPa (p = 0.046). The increase in the twinkling amplitude upon changing respiratory gas composition further supports the crevice microbubble hypothesis of twinkling. Higher pressures than explored in this study may be needed to reduce twinkling on in situ human kidney stones. [Work supported by the National Space Biomedical Research Institute through NASA NCC 9-58 and NIH grant DK043881.]

The effect of respiratory gas composition on kidney stone detection with the color Doppler ultrasound twinkling artifact

The color Doppler ultrasound twinkling artifact, which is thought to arise from microbubbles on and within the stone, has the potential to improve kidney stone detection in space; however, bubbles are known to be sensitive to the elevated levels of carbon dioxide (CO2) found on space vehicles. Here, we investigate the effect of respiratory gas composition on twinkling in swine implanted with kidney stones. Thirteen swine were initially exposed to either 100% oxygen (O2) or room air and then to air with elevated CO2 at 0.8%, 0.54%, or 0.27%. Stones were imaged with a Verasonics ultrasound system and ATL P4-2 transducer. The 9 swine initially breathing 100% O2 showed a significant reduction in twinkling when exposed to air with elevated CO2, with the degree of decrease in twinkling occurring in the order: 0.8%>0.54%>0.27% CO2. An additional 4 swine were oscillated between air with 0.04% CO2 (normal air) and 0.5% CO2. A reduction in twinkling was observed over the course of the experiment. The effect of respiratory gas composition should be further investigated before using twinkling to diagnose a kidney stone in space. [Work supported by NSBRI through NASA NCC 9-58 and NIH DK043881.]

The effect of ambient pressure on the color Doppler ultrasound twinkling artifact

Recently, our group discovered that overpressure suppressed the color Doppler ultrasound twinkling artifact on ex vivo calcium oxalate monohydrate (COM) kidney stones, suggesting that trapped microbubbles on the stone surface cause twinkling (Lu et al. 2013). Yet the hypothesis is not fully accepted, partly because bubbles were not observed. Here, we extend the overpressure results to include under-pressure and use high-speed photography to visualize the bubbles. A programmable ultrasound system with Philips/ATL P4-2 transducer was used. Ex vivo COM stones were placed in a hydraulic pressure chamber and imaged acoustically through an acrylic window. The overpressure threshold to diminish twinkling was found to vary significantly, with twinkling eliminated at pressures of 3 ATA (atmospheres absolute) up to &amp;gt;8 ATA, even within the same stone. When the stones were exposed to 0.2 ATA (under-pressure), twinkling increased. High-speed photography during Doppler ultrasound revealed only one instance of an oscillating bubble. However, when stones were exposed repeatedly to a pre-focal, off-axis lithotripter pulse (p+ = 1.5 MPa, p- = 2.5 MPa), stones that twinkled had bubbles emerge from the same location with each pulse whereas stones that did not twinkle had a random bubble distribution. [Work supported by NSBRI through NASA NCC 9-58 and NIH DK043881, DK092197.]