Whole Blood Viscosity Reference Intervals and Its Correlation with Hematology and Serum Chemistry in Cats Using Scanning Capillary Method.

Whole blood viscosity, a hemorheological factor, is currently used for diagnosis, as it is correlated with various vascular diseases that are difficult to diagnose early with a general blood test. It was determined that it was necessary to set reference intervals for further studies and utilization of whole blood viscosity in cats, a representative companion animal, and this study was conducted. Fifty healthy cats were recruited for the study, and whole blood viscosity, complete blood count, and serum chemistry tests were performed. The reference intervals of whole blood viscosity were 15.169 to 43.684 cP at a shear rate of 1 s-1 reflecting diastole, and 3.524 to 5.544 cP at a shear rate of 300 s-1 reflecting systole. Red blood cells, hematocrit, hemoglobin, white blood cells, and neutrophils in the complete blood count, and total protein, albumin, globulin, and cholesterol in the serum chemistry were significantly correlated with whole blood viscosity. The results of this study set the reference intervals of whole blood viscosity for healthy cats in a wide shear rate range that has not yet been fully established, and its correlation with other blood indicators investigated.

Alternative methods for calculating percentage haemolysis of red cell concentrates in peripheral blood banks in Sri Lanka.

Background: Haemolysis - one of the major limiting factors of red cell concentrate quality - must be measured as a quality-monitoring requirement. According to international quality standards, percentage haemolysis must be monitored in 1.0% of red cell concentrates produced monthly and maintained under 0.8%. Objective: This study assessed three alternative methods for determining plasma haemoglobin concentration in peripheral blood banks that lack a plasma or low haemoglobin photometer - the gold-standard method - in Sri Lanka. Methods: A standard haemolysate was prepared using an unexpired whole blood pack of normal haemoglobin concentration. A concentration series from 0.1 g/dL to 1.0 g/dL was prepared by diluting portions of standard haemolysate with saline. The alternative methods, namely visual haemoglobin colour scale, spectrophotometric calibration graph, and standard haemolysate capillary tube comparison, were designed using this concentration series and were used to test red cell concentrates received at the Quality Control Department of the National Blood Center, Sri Lanka, from February 2021 to May 2021. Results: A strong correlation was observed between the haemoglobin photometer method and the alternative methods (R = ~0.9). Based on the linear regression model, the standard haemolysate capillary tube comparison method was the best of the three alternative methods (R 2 = 0.974). Conclusion: All three alternative methods are recommended for use in peripheral blood banks. The standard haemolysate capillary tube comparison method was the best model.

An ultrasensitive detection platform for cocaine: Aptasensing strategy in capillary tube.

Cocaine as a detrimental addictive drug threats human health through inducing heart problem, blood pressure, anxiety, immunodeficiency, paranoia, and organ damage. Thus, the quantification of cocaine in the biological samples by a simple, high specificity, and fast method is highly urgent to decrease the harmful effect of the misuse of this drug. In this study, we constructed a novel fluorescent aptasensor by combining the fluorescein (FAM)-modified specific aptamer and AuNPs in a capillary tube as the sensing substrate for the first time. The presence of cocaine recovered the fluorescence response of the aptasensor through interaction with the aptamer and differentiation of the aptamer@AuNPs complex. By fluorescence microscopy imaging of the aptasensor substrate and its quantitative analysis, a remarkable linear range from 100 pM to 600 µM and the ultra-low limit of detection (LOD) as 0.31 pM were achieved for the target detection. Cocaine was successfully quantified in the real samples (human serum and urine) by using the aptasensor. The aptasensor is simple, easy-to-use, favorable applicability, and cost-effective; and to the best of our knowledge, it is the first use of the capillary tube as a sensing platform just by using about 3 µl of the samples. It is also an easy-to-carry tool, promising for the on-site target detection. Besides, it can be a portable device for monitoring cocaine by using a handheld single-beam fluorescence microscope. It can be an appropriate detection tool in forensic science and medicine.

Growth of Laser-Induced Microbubbles inside Capillary Tubes Affected by Gathered Light-Absorbing Particles.

Microbubbles have important applications in optofluidics. The generation and growth of microbubbles is a complicated process in microfluidic channels. In this paper, we use a laser to irradiate light-absorbing particles to generate microbubbles in capillary tubes and investigate the factors affecting microbubble size. The results show that the key factor is the total area of the light-absorbing particles gathered at the microbubble bottom. The larger the area of the particles at bottom, the larger the size of the microbubbles. Furthermore, the area is related to capillary tube diameter. The larger the diameter of the capillary tube, the more particles gathered at the bottom of the microbubbles. Numerical simulations show that the Marangoni convection is stronger in a capillary tube with a larger diameter, which can gather more particles than that in a capillary tube with a smaller diameter. The calculations show that the particles in contact with the microbubbles will be in a stable position due to the surface tension force.

Consecutive Sample Injection Analysis in Tube Radial Distribution Chromatography.

Tube radial distribution chromatography based on the tube radial distribution flow, or annular flow, in an open-tubular capillary has been reported, where the annular flow is created through phase-separation multiphase flow. We have proposed the first-ever procedure for consecutive sample injection analysis using chromatography. In basic terms, a commercially available HPLC system could be used with a sample injector (0.2 µL volume) and a fused-silica capillary tube (250 cm long) as a separation column instead of a normal packed one, while the built-in detection cell was replaced by improved on-capillary detection. A ternary mixed solution of water/acetonitrile/ethyl acetate (3:8:4 volume ratio) was delivered into the capillary tube as an eluent at a flow rate of 2.0 µL min-1. Model sample solutions of 1-naphthol and 2,6-naphthalenedisulfonic acid were consecutively analyzed by the present chromatography with a processing rate of 6 samples per hour. Simple and rapid consecutive analysis could be performed because washing and initialization of the separation tube was no longer necessary. The obtained results provide clues to developing new methodologies which combine features of both chromatography (separation) and the flow injection method (consecutive analysis).

Gold Nanoparticles Affect Pericyte Biology and Capillary Tube Formation.

Gold nanoparticles (AuNPs) are used for diagnostic and therapeutic purposes, especially antiangiogenesis, which are accomplished via inhibition of endothelial cell proliferation, migration, and tube formation. However, no research has been performed on the effects of AuNPs in pericytes, which play vital roles in endothelial cell functions and capillary tube formation during physiological and pathological processes. Therefore, the effects of AuNPs on the morphology and functions of pericytes need to be elucidated. This study treated human placental pericytes in monoculture with 20 nm AuNPs at a concentration of 30 ppm. Ki-67 and platelet-derived growth factor receptor-ß (PDGFR-ß) mRNA expression was measured using real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Cell migration was assessed by Transwell migration assay. The fine structures of pericytes were observed by transmission electron microscopy. In addition, 30 ppm AuNP-treated pericytes and intact human umbilical vein endothelial cells were cocultured on Matrigel to form three-dimensional (3D) capillary tubes. The results demonstrated that AuNPs significantly inhibited proliferation, reduced PDGFR-ß mRNA expression, and decreased migration in pericytes. Ultrastructural analysis of pericytes revealed AuNPs in late endosomes, autolysosomes, and mitochondria. Remarkably, many mitochondria were swollen or damaged. Additionally, capillary tube formation was reduced. We found that numerous pericytes on 3D capillary tubes were round and did not extend their processes along the tubes, which resulted in more incomplete tube formation in the treatment group compared with the control group. In summary, AuNPs can affect pericyte proliferation, PDGFR-ß mRNA expression, migration, morphology, and capillary tube formation. The findings highlight the possible application of AuNPs in pericyte-targeted therapy for antiangiogenesis.

Distance-Based Biosensor for Ultrasensitive Detection of Uracil-DNA Glycosylase Using Membrane Filtration of DNA Hydrogel.

In the deoxyribonucleic acid (DNA) repair pathways, DNA repair enzymes have great significance for genomic integrity. As one important initiator of the base-excision repair pathway, the aberrant activity of uracil-DNA glycosylase (UDG) is closely associated with many diseases. Herein, we developed a simple distance-based device for visual detection of UDG activity using a load-free DNA hydrogel. The DNA hydrogel consists of polyacrylamide-DNA chains being bridged by a single-stranded DNA crosslinker containing a responsive uracil base site. UDG can recognize and remove the uracil, resulting in the cleavage effect of the DNA crosslinker strand with the assistance of endonuclease IV (Endo IV). Plugging one end of the capillary tube, the DNA hydrogel acting as a filter membrane separator would control molecules to flow into the tube. The integrity of the DNA hydrogel networks is affected by the excision of UDG. Therefore, taking full advantage of membrane filtration of the DNA hydrogel, the activity of UDG can be quantitatively detected via reading the distance of the red indicator solution in the capillary tube. Without any instruments and complicated procedures, this method realizes high sensitivity and specificity for the detection of UDG as low as 0.02 mU/mL and can even measure UDG in complex cell samples. Additionally, this method is simple, universal, and can be used to screen inhibitors, which shows great potential for point-of-care testing, clinical diagnosis, and drug discovery.

Lab-On-Capillary Platform for On-Site Quantitative SERS Analysis of Surface Contaminants Based on Au@4-MBA@Ag Core-Shell Nanorods.

A portable and highly reproducible lab-on-capillary surface-enhanced Raman scattering (SERS) platform was developed using a specially designed homemade device for rapid on-site SERS measurement. In particular, this platform was composed of a capillary with a tiny orifice, which allows an effective and lossless sample extraction, resulting in high SERS performance. The capillary-based plasmonic substrate was prepared by compactly assembling Au@Ag core-shell nanorods (NRs) embedded with the 4-mercaptobenzoic acid (4-MBA) molecule as an internal standard onto the inner wall of a capillary tube. The fabrication process is facile and convenient with no requirement for complicated procedures. The exclusively prepared nanoparticles were able to significantly improve the signal consistency and overcome the limitations of reliable quantitative SERS analysis compared with conventional methods. Importantly, it was found that this capillary-based substrate with higher sensitivity was essentially attributed to more valid nanoparticles in the effective laser excitation region derived from the unique structure of the capillary. Furthermore, the applicability of the Au@4-MBA@Ag nanorod-decorated capillary for the quantitative identification of fungicides (malachite green and crystal violet) on the shell was demonstrated. As a result, this proposed lab-on-capillary sensor holds promising practical potential for rapid on-site analysis, especially for various contaminants on an uneven surface.

Deficiency of αII-spectrin affects endothelial cell-matrix contact and migration leading to impairment of angiogenesis in vitro.

BACKGROUND: Precise coordination of cytoskeletal components and dynamic control of cell adhesion and migration are required for crucial cell processes such as differentiation and morphogenesis. We investigated the potential involvement of αII-spectrin, a ubiquitous scaffolding element of the membrane skeleton, in the adhesion and angiogenesis mechanism. METHODS: The cell models were primary human umbilical vein endothelial cells (HUVECs) and a human dermal microvascular endothelial cell line (HMEC-1). After siRNA- and shRNA-mediated knockdown of αII-spectrin, we assessed its expression and that of its partners and adhesion proteins using western blotting. The phenotypes of the control and spectrin-depleted cells were examined using immunofluorescence and video microscopy. Capillary tube formation was assessed using the thick gel Matrigel matrix-based method and a microscope equipped with a thermostatic chamber and a Nikon Biostation System camera. RESULTS: Knockdown of αII-spectrin leads to: modified cell shape; actin cytoskeleton organization with the presence of peripheral actin patches; and decreased formation of stress fibers. Spectrin deficiency affects cell adhesion on laminin and fibronectin and cell motility. This included modification of the localization of adhesion molecules, such as αVß3- and α5-integrins, and organization of adhesion structures, such as focal points. Deficiency of αII-spectrin can also affect the complex mechanism of in vitro capillary tube formation, as demonstrated in a model of angiogenesis. Live imaging revealed that impairment of capillary tube assembly was mainly associated with a significant decrease in cell projection length and stability. αII-spectrin depletion is also associated with significantly decreased expression of three proteins involved in capillary tube formation and assembly: VE-cadherin, MCAM and ß3-integrin. CONCLUSION: Our data confirm the role of αII-spectrin in the control of cell adhesion and spreading. Moreover, our findings further support the participation of αII-spectrin in capillary tube formation in vitro through control of adhesion molecules, such as integrins. This indicates a new function of αII-spectrin in angiogenesis.

A Novel Improved Gram Staining Method Based on the Capillary Tube.

In this work, an exploratory study was conducted to examine Gram staining based on the capillary tube. Each Gram staining step for all bacterial strains tested was completed in capillary tubes. The results showed that different Gram staining morphologies were clearly visible in the capillary tubes. The results presented here demonstrated that the improved method could effectively distinguish between Gram-positive and Gram-negative bacteria, and only small volumes of reagents were required in this method. Collectively, this efficient method could rapidly and accurately identify the types of bacteria. Therefore, our findings could be used as a useful reference study for other staining methods.

MMP-9 Downregulation with Lipid Nanoparticles for Inhibiting Corneal Neovascularization by Gene Silencing.

Gene silencing targeting proangiogenic factors have been shown to be a useful strategy in the treatment of corneal neovascularization (CNV). Among interference RNA (RNAi) molecules, short-hairpin RNA (shRNA) is a plasmid-coded RNA able to down-regulate the expression of the desired gene. It is continuously produced in the host cell, inducing a durable gene silencing effect. The aim of this work was to develop a solid lipid nanoparticle (SLN)-based shRNA delivery system to downregulate metalloproteinase 9 (MMP-9), a proangiogenic factor, in corneal cells for the treatment of CNV associated with inflammation. The nanovectors were prepared using a solvent emulsification-evaporation technique, and after physicochemical evaluation, they were evaluated in different culture cell models. Transfection efficacy, cell internalization, cell viability, the effect on MMP-9 expression, and cell migration were evaluated in human corneal epithelial cells (HCE-2). The inhibition of tube formation using human umbilical vein endothelial cells (HUVEC) was also assayed. The non-viral vectors based on SLN were able to downregulate the MMP-9 expression in HCE-2 cells via gene silencing, and, consequently, to inhibit cell migration and tube formation. These results demonstrate the potential of lipid nanoparticles as gene delivery systems for the treatment of CNV-associated inflammation by RNAi technology.

Comparison of methodologies for detecting Trypanosoma cruzi parasites by microscopic observation of microhematocrit capillary tubes

Abstract INTRODUCTION: The microscopic examination of microhematocrit tubes (mHCT) has been proposed as the gold standard for acute and congenital Chagas disease diagnosis. We compared different mHCT methodologies detecting T. cruzi parasites in the blood. METHODS: The rotating method, water mount, and immersion oil methods were compared for their suitability, sensitivity, and specificity. RESULTS: The rotating method was easier, faster, and more sensitive than the others with 100% specificity. CONCLUSIONS: The rotating method is feasible for laboratory technicians with standard training in microscopic techniques and is recommended for the diagnosis of acute Chagas disease in primary health care facilities.

Tube radial distribution chromatography system developed by combining commercially available HPLC system and open-tubular capillary tube as separation column.

Tube radial distribution chromatography based on tube radial distribution flow, or annular flow, in an open-tubular capillary has been reported, where the annular flow is created through phase separation multiphase flow. The chromatographic system requires some specific instruments and treatments for microfluidic flow in the capillary tube. In this study, we developed a new set-up for tube radial distribution chromatography by combining a commercially available HPLC system with an open-tubular capillary tube (with an inner diameter of 100 µm) as a separation column instead of a conventional packed column. The analyte solution was injected with an injection valve (2 µL volume) and a ternary solution of water/acetonitrile/ethyl acetate (3:8:2 vol ratio) was delivered as the eluent to the capillary tube at a flow rate of 8.6 µL min-1. The chromatographic system, that is, the HPLC system equipped with the open-tubular capillary tube, could successfully separate the model analytes, 1-naphthol, 1-naphthalenesulfonic acid, and 2,6-naphthalenedisulfonic acid, with base-line separation. The inner and outer phases in the annular flow worked as the mobile and pseudo-stationary phases, respectively, in the tube radial distribution chromatography system. The experimentally obtained elution times of the analytes were compared with their corresponding theoretical values calculated using their capacity factors for the inner and outer phases and the linear flow velocities of the respective phases.

On-line Analysis of Catalytic Reaction Products Using a High-Pressure Tandem Micro-reactor GC/MS.

When a GC/MS system is coupled with a pressurized reactor, the separation efficiency and the retention time are directly affected by the reactor pressure. To keep the GC column flow rate constant irrespective of the reaction pressure, a restrictor capillary tube and an open split interface are attached between the GC injection port and the head of a GC separation column. The capability of the attached modules is demonstrated for the on-line GC/MS analysis of catalytic reaction products of a bio-oil model sample (guaiacol), produced under a pressure of 1 to 3 MPa.

Protein separation through preliminary experiments concerning pH and salt concentration by tube radial distribution chromatography based on phase separation multiphase flow using a polytetrafluoroethylene capillary tube.

Protein mixtures were separated using tube radial distribution chromatography (TRDC) in a polytetrafluoroethylene (PTFE) capillary (internal diameter=100µm) separation tube. Separation by TRDC is based on the annular flow in phase separation multiphase flow and features an open-tube capillary without the use of specific packing agents or application of high voltages. Preliminary experiments were conducted to examine the effects of pH and salt concentration on the phase diagram of the ternary mixed solvent solution of water-acetonitrile-ethyl acetate (8:2:1 volume ratio) and on the TRDC system using the ternary mixed solvent solution. A model protein mixture containing peroxidase, lysozyme, and bovine serum albumin was analyzed via TRDC with the ternary mixed solvent solution at various pH values, i.e., buffer-acetonitrile-ethyl acetate (8:2:1 volume ratio). Protein was separated on the chromatograms by the TRDC system, where the elution order was determined by the relation between the isoelectric points of protein and the pH values of the solvent solution.

Cost-Effective Plasmonic Platforms: Glass Capillaries Decorated with Ag@SiO2 Nanoparticles on Inner Walls as SERS Substrates.

A cost-effective method for the fabrication of a glass capillary based plasmonic platform for the selective detection and identification of analytes of importance in health, environment, and safety is demonstrated. This was achieved by coating Ag@SiO2 nanoparticles (Ag ∼ 60 nm) having silica shell of varying thickness (∼2 and ∼25 nm) on the inside walls of glass capillaries, over 2 cm in length, with uniform coverage. It was found that the particle density on the surface plays a decisive role on the enhancement of Raman signals. Multiple hot spots, which are essentially junctions of amplified electric field, were generated when ∼30 Ag@SiO2 particles/µm2 were bound onto the walls of glass capillaries. The pores of the silica shell allow the localization of analyte molecules to the vicinity of hot spots resulting in signal enhancements of the order of 1010 (using pyrene as analyte; excitation wavelength, 632.8 nm). The applicability of Ag@SiO2 coated capillaries for the detection of a wide range of molecules has been explored, by taking representative examples of polyaromatic hydrocarbons (pyrene), amino acids (tryptophan), proteins (bovine serum albumin), and explosives (trinitrotoluene). By increasing the thickness of the silica shell of Ag@SiO2 nanoparticles, an effective filtration cum detection method has been developed for the selective identification of small molecules such as amino acids, without the interference of large proteins.

A Low-Cost and Fast Real-Time PCR System Based on Capillary Convection.

A low-cost and fast real-time PCR system in a pseudo-isothermal manner with disposable capillary tubes based on thermal convection for point-of-care diagnostics is developed and tested. Once stable temperature gradient along the capillary tube has been established, a continuous circulatory flow or thermal convection inside the capillary tube will repeatedly transport PCR reagents through temperature zones associated with the DNA denaturing, annealing, and extension stages of the reaction. To establish stable temperature gradient along the capillary tube, a dual-temperature heating strategy with top and bottom heaters is adopted here. A thermal waveguide is adopted for precise maintenance of the temperature of the top heater. An optimized optical network is developed for monitoring up to eight amplification units for real-time fluorescence detection. The system performance was demonstrated with repeatable detection of influenza A (H1N1) virus nucleic acid targets with a limit of detection of 1.0 TCID50/mL within 30 min.

Rhipicephalus (Boophilus) microplus tick in vitro feeding methods for functional (dsRNA) and vaccine candidate (antibody) screening.

Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) ticks cause economic losses for cattle industries throughout tropical and subtropical regions of the world estimated at $US2.5 billion annually. Lack of access to efficacious long-lasting vaccination regimes and increases in tick acaricide resistance have led to the investigation of targets for the development of novel tick vaccines and treatments. In vitro tick feeding has been used for many tick species to study the effect of new acaricides on the transmission of tick-borne pathogens. Few studies have reported the use of in vitro feeding for functional genomic studies using RNA interference and/or the effect of specific anti-tick antibodies. In particular, in vitro feeding reports for the cattle tick are limited due to its relatively short hypostome. Previously published methods were further modified to broaden optimal tick sizes/weights, feeding sources including bovine and ovine serum, optimisation of commercially available blood anti-coagulant tubes, and IgG concentrations for effective antibody delivery. Ticks are fed overnight and monitored for ∼5-6 weeks to determine egg output and success of larval emergence using a humidified incubator. Lithium-heparin blood tubes provided the most reliable anti-coagulant for bovine blood feeding compared with commercial citrated (CPDA) and EDTA tubes. Although >30mg semi-engorged ticks fed more reliably, ticks as small as 15mg also fed to repletion to lay viable eggs. Ticks which gained less than ∼10mg during in vitro feeding typically did not lay eggs. One mg/ml IgG from Bm86-vaccinated cattle produced a potent anti-tick effect in vitro (83% efficacy) similar to that observed in vivo. Alternatively, feeding of dsRNA targeting Bm86 did not demonstrate anti-tick effects (11% efficacy) compared with the potent effects of ubiquitin dsRNA. This study optimises R. microplus tick in vitro feeding methods which support the development of cattle tick vaccines and treatments.

Reference intervals for whole blood viscosity using the analytical performance-evaluated scanning capillary tube viscometer.

OBJECTIVES: This study was performed to establish the reference intervals for whole blood viscosity (WBV) using the analytical performance-evaluated scanning capillary tube viscometer (SCTV). DESIGN AND METHODS: The analytical performance of the SCTV was evaluated using three different levels of QC materials and sixty human EDTA-blood samples. To establish the reference intervals for WBV, 297 healthy individuals (123 men and 174 women) were selected from 1083 subjects. RESULTS: Within-day precisions with QC materials and human whole blood and between-day precisions with QC materials were below 5.0%, 6.6% and 8.0% in CVs at all shear rates, respectively. Comparison tests between the SCTV and the Brookfield viscometer showed a significant correlation (R(2)=0.972, p<0.001). The reference intervals for WBV in healthy men were 3.66-5.41cP at 300s(-1) and 23.15-36.45cP at 1s(-1) while those in women were 3.27-4.32cP at 300s(-1) and 18.20-27.36cP at 1s(-1), respectively. CONCLUSIONS: Using the analytical performance-evaluated SCTV, the reference intervals for WBV were established in healthy adults, which could be beneficial to the clinical utility of WBV in the aspect of appropriate modalities for the improvement of blood viscosity.

A technique for evaluating the oil/heavy-oil viscosity changes under ultrasound in a simulated porous medium.

Theoretically, Ultrasound method is an economical and environmentally friendly or "green" technology, which has been of interest for more than six decades for the purpose of enhancement of oil/heavy-oil production. However, in spite of many studies, questions about the effective mechanisms causing increase in oil recovery still existed. In addition, the majority of the mechanisms mentioned in the previous studies are theoretical or speculative. One of the changes that could be recognized in the fluid properties is viscosity reduction due to radiation of ultrasound waves. In this study, a technique was developed to investigate directly the effect of ultrasonic waves (different frequencies of 25, 40, 68 kHz and powers of 100, 250, 500 W) on viscosity changes of three types of oil (Paraffin oil, Synthetic oil, and Kerosene) and a Brine sample. The viscosity calculations in the smooth capillary tube were based on the mathematical models developed from the Poiseuille's equation. The experiments were carried out for uncontrolled and controlled temperature conditions. It was observed that the viscosity of all the liquids was decreased under ultrasound in all the experiments. This reduction was more significant for uncontrolled temperature condition cases. However, the reduction in viscosity under ultrasound was higher for lighter liquids compare to heavier ones. Pressure difference was diminished by decreasing in the fluid viscosity in all the cases which increases fluid flow ability, which in turn aids to higher oil recovery in enhanced oil recovery (EOR) operations. Higher ultrasound power showed higher liquid viscosity reduction in all the cases. Higher ultrasound frequency revealed higher and lower viscosity reduction for uncontrolled and controlled temperature condition experiments, respectively. In other words, the reduction in viscosity was inversely proportional to increasing the frequency in temperature controlled experiments. It was concluded that cavitation, heat generation, and viscosity reduction are three of the promising mechanisms causing increase in oil recovery under ultrasound.