Transthoracic echocardiography for imaging of the different coronary artery segments: a feasibility study

Background

Cardiac resynchronisation therapy (CRT) improves mortality and morbidity in heart failure patients with wide QRS. Observational studies suggest that patients having more left ventricular dyssynchrony pre-implantation obtain greater benefit on ventricular function and symptoms with CRT.

Aim

To provide an analysis of the prevalence and type of dyssynchrony in patients included in the CARE-HF trial.

Methods

100 patients 67 (58 to 71) years were examined with echocardiography including tissue doppler imaging before receiving a CRT-pacemaker. Atrio-ventricular dyssynchrony (LVFT/RR) was defined as left ventricular filling time <40% of the RR-interval. Inter-ventricular mechanical delay (IVMD) was measured as the difference in onset of Doppler-flow in the pulmonary and aortic outflow tracts >40 ms. Intra-ventricular (regional) dyssynchrony in a 16-segment model was expressed either as a delayed longitudinal contraction (DLC) during the postsystolic phase or by tissue synchronisation imaging (TSI) with a predefined time-difference in systolic maximal velocities >85 ms.

Results

LVFT/RR was present in 34% and IVMD in 60% of patients while intra-ventricular dyssynchrony was present in 85% (DLC) and 86% (TSI) with a high agreement between the measures (Kappascore 0.86–1.00), indicating the methods being interchangeable. Patients with cardiomyopathy (53%) were more likely to have LVFT/RR <40% (45% vs. 21% (p= 0.02)) and more segments affected by intra-ventricular dyssynchrony 4(3, 5) vs. 3(1, 4), p = 0.002, compared to patients with ischemic heart disease.

Conclusion

The prevalence of intra-ventricular dyssynchrony is high in patients with heart failure, wide QRS and depressed systolic function. Most important, TSI appears to be a fast and reliable method to identify patients with intra-ventricular dyssynchrony likely to benefit from CRT.     

Dynamic imaging of the immune system: progress, pitfalls and promise

Both innate and adaptive immunity are dependent on the migratory capacity of myeloid and lymphoid cells. Effector cells of the innate immune system rapidly enter infected tissues, whereas sentinel dendritic cells in these sites mobilize and transit to lymph nodes. In these and other secondary lymphoid tissues, interactions among various cell types promote adaptive humoral and cell-mediated immune responses. Recent advances in light microscopy have allowed direct visualization of these events in living animals and tissue explants, which allows a new appreciation of the dynamics of immune-cell behaviour. In this article, we review the basic techniques and the tools used for in situ imaging, as well as the limitations and potential artefacts of these methods.     

Contrast-enhanced MRA of abdomen and lower limbs arteries: artifacts and pitfalls

The purpose of the work was the analysis of the most widespread diagnostic mistakes in connection with the technical artifacts arising at performing contrast-enhanced MR-angiography of abdomen and lower limbs arteries.     

Telomere length measurement by fluorescence in situ hybridization and flow cytometry: tips and pitfalls

BACKGROUND: Telomeres containing noncoding DNA repeats at the end of the chromosomes are essential for chromosomal stability and are implicated in regulating the replication and senescence of cells. The gradual loss of telomere repeats in cells has been linked to aging and tumor development and methods to measure telomere length are of increasing interest. At least three methods for measuring the length of telomere repeats have been described: Southern blot analysis and quantitative fluorescence in situ hybridization using either digital fluorescence microscopy (Q-FISH) or flow cytometry (flow-FISH). Both Southern blot analysis and Q-FISH have specific limitations and are time-consuming, whereas the flow-FISH technique requires relatively few cells (10(5)) and can be completed in a single day. A further advantage of the flow-FISH method is that data on the telomere length from individual cells and subsets of cells (lymphocytes and granulocytes) can be acquired from the same sample. In order to obtain accurate and reproducible results using the flow-FISH technique, we systematically explored the influence of various steps in the protocol on telomere length values and established an acceptable range for the most critical parameters. METHODS: Isolated leukocytes from whole blood are denatured by heat and 70%/75% formamide, then hybridized with or without a telomere-specific fluorescein isothiocyante (FITC)-conjugated peptide nucleic acid probe (PNA). Unbound telomere PNA is washed away, the DNA is counterstained, and telomere fluorescence is measured on a flow cytometer using an argon ion laser (488 nm) to excite FITC. For each sample, duplicates of telomere PNA-stained and unstained tubes are analyzed. RESULTS: Cell counts and flow-FISH telomere length measurements were performed on leukocytes and thymocytes of humans and other species. Leukocyte suspensions were prepared by two red blood cell lysis steps with ammonium chloride. Optimal denaturation of DNA was achieved by heating at 85-87 degrees C for 15 min in a solution containing 70%/75% formamide. Hybridization was performed at room temperature with a 0.3 microg/ml telomere-PNA probe for at least 60-90 min. Unbound telomere-PNA probe was diluted at least 4,000-40,000 times with wash steps containing 70%/75% formamide at room temperature. LDS 751 and DAPI were suitable as DNA counterstains as they did not show significant interference with telomere length measurement. CONCLUSIONS: The use of flow-FISH for telomere length measurements in nucleated blood cells requires tight adherence to an optimized protocol. The method described here can be used to determine rapidly the telomere length in subsets of nucleated blood cells.     

Confocal imaging of metabolism in vivo: pitfalls and possibilities

Confocal laser scanning microscopy (CLSM) has had wide application in morphological studies and ion imaging in plants, but little impact so far on biochemical investigations. This position is likely to change as the range of fluorescent probes increases. To illustrate the type of kinetic information that can be obtained using CLSM in an intact, living system, an analysis has been made of the two-step detoxification of monochlorobimane (MCB) following conjugation to glutathione (GSH) by a glutathione S-transferase in the cytoplasm and vacuolar sequestration of the fluorescent glutathione S-bimane (GSB) by a glutathione S-conjugate (GSX) pump. Fluorescence from the cytoplasm and vacuole of individual trichoblasts and atrichoblasts was measured from time-series of (x, y) optical sections in the elongation zone of Arabidopsis root tips. Intensity changes were calibrated and converted to amounts using compartment volumes, measured by stereological techniques. The data were well described using pseudo-first-order kinetics for the conjugation reaction and either Michaelis-Menten kinetics (Model I), or, as the GSX-pump was operating close to V(max), a pseudo-zero-order reaction (Model II), for the GSX-pump. Analysis of 15 individual cells from two roots gave [GSH](cyt) in the range 1.8-4 mM. GST activity was relatively constant on a cell basis in one root, but increased markedly in the other, giving a net increase in conjugation activity as cells progressed through the elongation zone. In contrast, GSX-pump activity increased in parallel with the increase in cell size in both roots, effectively maintaining a constant transport activity per unit root length or estimated vacuole surface area.     

Prinzmetal's angina,normal coronary arteries and pericarditis.

A woman with Prinzmetal''s variant angina had spontaneous attacks of myocardial ischemia characterized by severe chest pain, hypotension, inferior ST-segment elevation, transient complete heart block and selective right ventricular dysfunction. Despite initial improvement following intravenous administration of atropine and sublingual administration of nitroglycerin she died of cardiogenic shock. Autopsy showed normal coronary arteries and acute pericarditis, more pronounced over the right side of the heart. It is postulated that the pericardial inflammation elicited severe spasm of the subjacent right coronary artery.     

Real-time ultrasonic imaging of the peripheral arteries: Technique, normal anatomy, and pathology

Real-time ultrasonic echo imaging of peripheral arteries promises to facilitate the management of selected patients with peripheral and extracranial arterial disease. This report outlines the technique of imaging the carotid system and portions of the arteries that supply the lower extremities. It also discusses the normal and pathologic anatomy of these arteries.     

Myocardial infarction,hyperthyroidism and normal coronary arteries: report of two cases.

Myocardial infarction is uncommon in persons with hyperthyroidism and also uncommon in the absence of demonstrable coronary artery disease. Cardiac catheterization and selective coronary angiography were performed in two men following apparent myocardial infarctions. Both patients were 33 years of age, thyrotoxic and angiographically free of coronary artery abnormalities.     

Adrenergic control of coronary arteries

This review of adrenergic control of coronary arteries is based on studies conducted on isolated vessels. Both alpha- and beta-adrenergic receptors are present in the coronary vasculature but with different distributions. The large coronary arteries have a larger percentage of alpha receptors, which mediate contraction, whereas the small coronary arteries are equipped almost exclusively with beta receptors, which mediate relaxation. The beta receptors are of the subclass beta 1. Both alpha 1 and alpha 2 receptors are present in the large coronaries. The alpha 2 receptors are prejunctional and inhibit neuronal release of norepinephrine (NE), and are also postjunctional and mediate contraction of the vascular smooth muscle. Evidence is reviewed suggesting that coronary alpha and beta receptors may be different conformational states of the same parent macromolecule in which the alpha state predominates at lower temperatures. Contrary to these results we have observed that whereas the response of the large coronary artery to NE at 39 C is predominantly constriction, it is predominantly dilation at 29 C. The characteristic of the adrenergic receptor is clearly temperature dependent.     

Assessing insect biodiversity with automatic light traps in Brazil: Pearls and pitfalls of metabarcoding samples in preservative ethanol

Automated species identification based on data produced with metabarcoding offers an alternative for assessing biodiversity of bulk insect samples obtained with traps. We used a standard two‐step PCR approach to amplify a 313 bp fragment of the barcoding region of the mitochondrial COI gene. The PCR products were sequenced on an Illumina MiSeq platform, and the OTUs production and taxonomic identifications were performed with a customized pipeline and database. The DNA used in the PCR procedures was extracted directly from the preservative ethanol of bulk insect samples obtained with automatic light traps in 12 sampling areas located in different biomes of Brazil, during wet and dry seasons. Agricultural field and forest edge habitats were collected for all sampling areas. A total of 119 insect OTUs and nine additional OTUs assigned to other arthropod taxa were obtained at a ≥97% sequence similarity level. The alpha and beta diversity analyses comparing biomes, habitats, and seasons were mostly inconclusive, except for a significant difference in beta diversity between biomes. In this study, we were able to metabarcode and HTS adult insects from their preservative medium. Notwithstanding, our results underrepresent the true magnitude of insect diversity expected from samples obtained with automatic light traps in Brazil. Although biological and technical factors might have impacted our results, measures to optimize and standardize eDNA HTS should be in place to improve taxonomic coverage of samples of unknown diversity and stored in suboptimal conditions, which is the case of most eDNA samples.     

Current state of whole slide imaging use in cytopathology: Pros and pitfalls

Whole slide imaging (WSI) allows generation of large whole slide images and their navigation with zoom in and out like a true virtual microscope. It has become widely used in surgical pathology for many purposes, such as education and training, research activity, teleconsultation, and primary diagnosis. However, in cytopathology, the use of WSI has been lagging behind histology, mainly due to the cytological specimen's characteristics, as groups of cells of different thickness are distributed throughout the slide. To allow the same focusing capability of light microscope, slides have to be scanned at multiple focal planes, at the cost of longer scan times and larger file size. These are the main technical pitfalls of WSI for cytopathology, partly overcome by solutions like liquid‐based preparations. Validation studies for the use in primary diagnosis are less numerous and more heterogeneous than in surgical pathology. WSI has been proved effective for training students and successfully used in proficiency testing, allowing the creation of digital cytology atlases. Longer scan times are also a barrier for use in rapid on‐site evaluation, but WSI retains its advantages of easy sharing of images for consultation, multiple simultaneous viewing in different locations, the possibility of unlimited annotations and easy integration with medical records. Moreover, digital slides set the laboratory free from reliance on a physical glass slide, with no more concern of fading of stain or slide breakage. Costs are still a problem for small institutions, but WSI can also represent the beginning of a more efficient way of working.     

Spectroscopic imaging of arteries and atherosclerotic plaques

Fourier transform infrared (FTIR) spectroscopic imaging using a focal plane array detector has been used to study atherosclerotic arteries with a spatial resolution of 3-4 microm, i.e., at a level that is comparable with cellular dimensions. Such high spatial resolution is made possible using a micro-attenuated total reflection (ATR) germanium objective with a high refractive index and therefore high numerical aperture. This micro-ATR approach has enabled small structures within the vessel wall to be imaged for the first time by FTIR. Structures observed include the elastic lamellae of the tunica media and a heterogeneous distribution of small clusters of cholesterol esters within an atherosclerotic lesion, which may correspond to foam cells. A macro-ATR imaging method was also applied, which involves the use of a diamond macro-ATR accessory. This study of atherosclerosis is presented as an illustrative example of the wider potential of these ATR imaging approaches for cardiovascular medicine and biomedical applications.     

Leptin receptors are expressed in coronary arteries, and hyperleptinemia causes significant coronary endothelial dysfunction

Obesity is associated with marked increases in plasma leptin concentration, and hyperleptinemia is an independent risk factor for coronary artery disease. As a result, the purpose of this investigation was to test the following hypotheses: 1) leptin receptors are expressed in coronary endothelial cells; and 2) hyperleptinemia induces coronary endothelial dysfunction. RT-PCR analysis revealed that the leptin receptor gene is expressed in canine coronary arteries and human coronary endothelium. Furthermore, immunocytochemistry demonstrated that the long-form leptin receptor protein (ObRb) is present in human coronary endothelium. The functional effects of leptin were determined using pressurized coronary arterioles (<130 microm) isolated from Wistar rats, Zucker rats, and mongrel dogs. Leptin induced pharmacological vasodilation that was abolished by denudation and the nitric oxide synthase inhibitor N(omega)-nitro-l-arginine methyl ester and was absent in obese Zucker rats. Intracoronary leptin dose-response experiments were conducted in anesthetized dogs. Normal and obese concentrations of leptin (0.1-3.0 microg/min ic) did not significantly change coronary blood flow or myocardial oxygen consumption; however, obese concentrations of leptin significantly attenuated the dilation to graded intracoronary doses of acetylcholine (0.3-30.0 microg/min). Additional experiments were performed in canine coronary rings, and relaxation to acetylcholine (6.25 nmol/l-6.25 micromol/l) was significantly attenuated by obese concentrations of leptin (625 pmol/l) but not by physiological concentrations of leptin (250 pmol/l). The major findings of this investigation were as follows: 1) the ObRb is present in coronary arteries and coupled to pharmacological, nitric oxide-dependent vasodilation; and 2) hyperleptinemia produces significant coronary endothelial dysfunction.     

Silicone breast implant rupture: pitfalls of magnetic resonance imaging and relative efficacies of magnetic resonance, mammography, and ultrasound

The objective of this study was to evaluate the relative efficacies of magnetic resonance (MR) imaging, ultrasonography, and mammography in implant rupture detection and to illustrate pitfalls in MR image interpretation. Thirty patients referred by plastic surgeons with suspected breast implant rupture were prospectively evaluated using MR, ultrasonography, and mammography. Imaging examinations were interpreted independently and blindly for implant rupture and correlated to operative findings. Surgical correlation in 16 patients (53 percent) with 31 implants showed 13 (42 percent) were intact, 5 (16 percent) had severe gel bleed, and 13 (42 percent) were ruptured. MR sensitivity was 100 percent and specificity was 63 percent. Accuracy for rupture was 81 percent with MR, higher than with ultrasonography and mammography (77 and 59 percent, respectively). We describe a specific pitfall in MR interpretation, the "rat-tail" sign, composed of a medial linear extension of silicone along the chest wall. Seen in eight cases (four intact, three ruptures, one gel bleed), the rat-tail sign may lead to misdiagnosis of implant rupture if seen in isolation. Magnetic resonance imaging is more accurate and sensitive than ultrasonography and mammography in detecting breast implant rupture. We describe a new sign (rat-tail sign) composed of medial compression of the implant simulating silicone extrusion as a potential false-positive MR finding for rupture. This article presents clinical experience with magnetic resonance, mammography, and ultrasound in the diagnosis of implant rupture and defines and illustrates potential pitfalls of MR interpretation, including the new rat-tail sign.     

Potentials and pitfalls of fluorescent quantum dots for biological imaging

Fluorescent semiconductor nanocrystals, known as quantum dots (QDs), have several unique optical and chemical features. These features make them desirable fluorescent tags for cell and developmental biological applications that require long-term, multi-target and highly sensitive imaging. The improved synthesis of water-stable QDs, the development of approaches to label cells efficiently with QDs, and improvements in conjugating QDs to specific biomolecules have triggered the recent explosion in their use in biological imaging. Although there have been many successes in using QDs for biological applications, limitations remain that must be overcome before these powerful tools can be used routinely by biologists.     

Branching characteristics of human coronary arteries

Branching angles and branch diameters were measured in a total of 850 arterial junctions in the coronary networks of two human hearts. Comparison is made with similar data obtained previously from the coronary networks of rats, and with what is considered to be optimum on theoretical grounds. It is concluded that the branching characteristics of the human coronary arteries are closer to the theoretical optimum than those of the coronary networks of rats. While the human data exhibit some departure from optimality and a good amount of scatter, these are well within levels observed elsewhere in the cardiovascular systems of man and animals, and considerably better than those found in the coronary networks of rats. The departure from optimality, in terms of physiological cost to the system, is within 5% for most data points.