Ultrasound contrast agents: basic principles.

INTRODUCTION: Ultrasonography lacked substances to be administered to patients to improve or increase the diagnostic yield, which is peculiar considering that contrast agents have long been used with all the other imaging techniques. Fortunately some contrast agents, most of them consisting in gas microbubbles, have been recently introduced for ultrasound imaging too: this review will focus on their history, behavior, current applications and future developments. Echocontrast agent research is in progress and many new agents are expected to be marketed this and next year, to be added to Levovist by Schering AG (Berlin, Germany), to enhance the ultrasound signal safely and effectively. No definitive conclusions can be drawn yet on the actual merits of each contrast agent, but all of them seem to be both effective and safe, meaning that their future success will depend on the relative cost-effectiveness and peculiarities. THE BASIC PRINCIPLES OF ECHOCONTRAST AGENTS: The microbubbles act as echo-enhancers by basically the same mechanism as that determining echo-scattering in all the other cases of diagnostic ultrasound, namely that the backscattering echo intensity is proportional to the change in acoustic impedance between the blood and the gas making the bubbles. The different acoustic impedance at this interface is very high and in fact all of the incident sound is reflected, even though not all of it will of course go back to the transducer. But the acoustic wave reflection, though nearly complete, would not be sufficient to determine a strong US enhancement because the microbubbles are very small and are sparse in the circulation. Moreover, reflectivity is proportional to the fourth power of a particle diameter but also directly proportional to the concentration of the particles themselves. SECOND HARMONIC IMAGING: As we said above, the microbubbles reached by an ultrasound signal resonate with a specific frequency depending on microbubble diameter. However, the main resonance frequency is not the only resonance frequency of the bubble itself and multiple frequencies of the fundamental one are emitted, just like in a musical instrument. These harmonic frequencies have decreasing intensity, but the second frequency, known as the second harmonic, is still strong enough to be used for diagnostic purposes. The theoretical advantage of the harmonic over the fundamental frequency is that only contrast agent microbubbles resonate with harmonic frequencies, while adjacent tissues do not resonate, or else their harmonic resonation is very little. Thus, using a unit especially set to produce ultrasounds at a given frequency (3.5 MHz) and receive an ultrasound signal twice as powerful (7 MHz) it will be possible to show the contrast agent only, without any artifact from the surrounding anatomical structures, with a markedly improved signal-to-noise ratio. A similar effect to digital subtraction in angiography can thus be obtained, even though through a totally different process. Moreover, second harmonic imaging permits to show extremely small vessels (down to 40 microm) with very slow flow, which would be missed with a conventional method. B-mode imaging can also depict the microbubbles in the myocardium suppressing nearly all the artifacts from cardiac muscle motion. Recently a peculiar behavior of microbubbles has been observed which may permit contrast agent detection even in capillaries. This method is variously known as sonoscintigraphy, loss of correlation, stimulated acoustic emission and transient scattering. The contrast agent microbubbles reached by an ultrasound beam powerful enough explode producing a strong and very short backscatter echo which is read by the unit as a Doppler signal and results in a color pixel where the individual microbubble exploded. CONCLUSIONS: The microbubble contrast agents developed and introduced as safe and effective echo-enhancers in present-day clinical practice will open up new oppurtunities

Contrast enhancing agents in ultrasonography: clinical applications.

INTRODUCTION: As ultrasound remains a poorly sensitive method, echocontrast agents make a real difference. At least 29 echocontrast agents are currently on trial worldwide; their chemical composition, mechanisms of action and possible clinical applications are different. The state of the art of echocontrast agents is discussed: their established applications, those expected in the near future and finally their hypothetical, ideal applications. POTENTIAL CLINICAL APPLICATIONS: An extravascular and a vascular domain can be considered. The former includes the gastrointestinal tract and body cavities--both the normal (bladder, uterus, tubes and so on) and the abnormal (abscesses, fistulas, pericardium, peritoneum and so on) ones. Echocontrast agents can: (1) create or improve an acoustic window; (2) distend some organs and fill them with a liquid, with homogenous attenuation of the ultrasound beam; (3) displace the air-containing intestinal loops; (4) depict the walls, the shape and the contours of a normal or abnormal cavity; (5) detect abnormal communications, fistulas and drainages; and (6) evaluate the amount of fluid in the pleural, pericardial or peritoneal cavities. As for vascular applications, this domain sees the highest number of echocontrast agents on trial or on the market. The best know of them are: Levovist (Schering AG, Berlin, Germany), BR1 (Bracco, Milan, Italy) and EchoGen (Abbott, USA). All these act by enhancing arteries, veins and capillaries. The clinical applications validated in clinical trials mainly regard studies in intracranial and neck vessels and the vascularity of upper and especially lower limbs of renal vessels. Tumor macrovascularity (and in the future, hopefully microvascularity) can also be studied in parenchymatous and/or glandular organs, as well as in intra- and extra-abdominal parenchymatous organs in the periskeletal soft tissues. Clinical validation has also been obtained in the follow-up of tumors submitted to ablation therapy (chemoembolization, ethanol injection, thermal ablation) and in echocardiography, both for morphological studies in the cardiac cavities and for the cardiac wall perfusion. CONCLUSIONS: In a subgroup of 513 out of 1275 patients examined Europe-wide, the contrast agent Levovist increased the diagnostic confidence from 27.4 +/- 22.5 to 77.2 +/- 22.5%. Such data encourage further trials to validate current preliminary results.

Control through breeding methods of factors affecting nutritional quality of cereals and grain legumes.

The comparison of nutritional quality parameters of proteins from cultivated cereal and legume species with animal proteins indicate the poor nutritional value of these plant products. The nutritive value of different cereal and legume species is very variable and large differences have also been observed coming from cultivars belonging to the same species. Many interpreting factors, such as protein content, essential amino acid composition and availability, protein digestibility and others are involved in such a highly variability. In addition, cereals as well as legumes may contain large amounts of antinutritional factors which can have serious effects under particular circumstances (e.e. tannins in a low-protein diet or phytates in a metal-deficient diet). Some legume species also contain chemicals of a different nature (i. e. lathyrogens, cyanogenetic glycosides, and others) which may be extremely toxic when ingested in significant amounts. The plant breeder attempting to develop higher-yielding, disease-resistant and nutritionally-improved crop varieties should be aware of such a complex of factors and alert to the possible production or increase of undesirable products or deleterious changes in chemical composition. Available data, although rather limited, indicate valuable breeding approaches to the improvement of nutritive value of cereal and legume grains for humans.

Contrast enhanced ultrasound of breast cancer.

The importance of ultrasound examination in the diagnosis of breast cancer has been widely demonstrated. During the last few years, the introduction of ultrasound contrast media has been considered a promising tool for studying the vascular pattern of focal lesions within the breast. Our purpose was to assess whether contrast-enhanced (CE) ultrasound examination, performed using specific contrast imaging modes, can be helpful for detection and characterization of breast lesions, and for prediction of the response of breast cancer to therapy.

Breeding possibilities offered by induced mutations in durum wheat.

A report on the use of mutations of agronomic value obtained in durum wheat in an accelerated crossing programme is given. The mutated characters used were isolated in cv. Cappelli and Russello, related respectively to a dominant short straw and a dominant earliness factor. Besides these two mutant lines, 4 other varieties were used, 80/57, Yuma, LD 357 and Kyperounda, representative of cultivated types of quite different origin. The program started in December 1965 and progressed till June 1970. During this time nine generations were grown, including two agronomic trials. Single crosses were also compared with incomplete backcrossing (3 backcrossing cycles), dominance of the factors utilized allowing backcrossing of every F1.

Genetic analysis of tetraploid and hexaploid wheat by utilization of monopentaploid hybrids.

This report deals with a method of analysis which uses existing hexaploid wheat monosomics to establish gene-chromosome associations in a tetraploid variety. Monosomics of Triticum aestivum cv. Chinese Spring belonging to the 14 lines of A and B genomes were crossed as female parents with Triticum durum cv. Capeiti, a spring type at present widely grown in Italy. For each line, two F 1 populations were obtained, normal pentaploids (2 n = 35) and monopentaploid (2 n = 34), in which, in turn, the monosomic A or B chromosome present was supplied by the tetraploid wheat. The morphological and physiological differences observed in the monopentaploid lines are attributed to differential expression of the genetic information concerning the character investigated, carried by the chromosome present in hemizygous condition. Then, only recessive or partially dominant alleles of the variety to be tested can be identified and attributed to a specific chromosome in the F 1 generation.Eight parameters were analyzed: culm and spike length, length and width of 1st (flag) and 2nd uppermost leaves, days from germination to heading and awn development.As far as culm length is concerned, although heterotic effect is present, seven chromosomes seem to be responsible for the modification of this character (1A, 2A, 2B, 3B, 4B, 5B, and 6 A); chromosomes 2A and 2B in particular, carry major factor (s) for plant height. A similar picture is presented by spike length which seems to be controlled by factors located in several chromosomes belonging to homoeologous groups 1, 2, 3 and 5, as well as the chromosome 4B.Leaf length, also, shows a complex pattern of inheritance. Monosomic conditions for chromosomes 1A and 1B increased, while monosomy for 5A and 5B significantly decreased, leaf length. A highly significant correlation was found between the mean lengths of the 1st and 2 nd leaves (= 0.74). Some monosomic lines (4A, 4B, 5A; 5B; 6A; 7A and 7B) had leaves significantly narrower than in the control and only monosomic 2A had broader leaves. The period from germination to heading seems to be influenced by at least 6 chromosomes. Three monosomic lines are significantly earlier (mono 1A, 7A and 5B) and three (mono 5A, 2B and 7B) are significantly later than the hybrid control.Finally, 8 monosomic lines were found to interfere significantly with awn development. Three lines (mono 2A, 2B and 7A) show a decrease and 5 (mono 1B; 3A, 3B; 4B and 6B) show an increase in awn development. On the basis of evidence in the literature and our own results, it appears that this analysis fits previous results perfectly and actually adds to the picture two further awn-promoting factors, A9 and A10, located on the 7A and 1B chromosomes respectively.

Genetic control of esterases in common wheat.

A refined technique of gel electrofocusing revealed the esterases in caryopses of Triticum aestivum, T. durum, and Triticum timopheevi. In T. aestivum, 17 isoenzymatic bands were ascertained in the pH 5-8 range: 11 were of higher intensity, 4 were weak and two very weak. Using Chinese Spring nulli-tetrasomic lines it was possible to locate the genetic control of several isoenzymes in the chromosomes of the homoeologous group 3. In chromosome 3A three bands are coded; in 3B four bands are coded; and in 3D two bands out of the eleven of higher intensity. T. durum, as expected, lacks bands coded in T. aestivum by chromosome 3D. T. timopheevi presents a quite distinct isoenzyme pattern, thus confirming its different speciation. Two major bands do not disappear in any of the nulli-tetra lines analyzed: it is supposed that these isoenzymes could be coded by at least two of the chromosomes of the group three involved in esterase control. The presence of several esterase isoenzymes in wheat is both evidence of their additivity with increasing ploidy level and biochemical support for the hypothesis that there is a higher possibility of adaptation of polyploids compared with diploid species.

An immunochemical approach to species relationship in Triticum and some related species.

An immunological reaction, precipitation in gel, was produced using a rabbit antiserum directed to a specific protein constantly present in bread wheats (T. aestivum, genome AABBDD), but absent in durum wheat (T. durum Desf., genome AABB). This protein was isolated in the soluble-protein fraction of bread wheat caryopses by combined biochemical and immunological techniques.The availability of such a specific anti-bread wheat serum made possible the analysis of a series of varieties and species of wheat and of some closely related (Secale, Aegilops) and less closely related (Hordeum, Haynaldia) taxa to determine whether the protein was present or absent. Hordeum vulgare, Haynaldia villosa, Triticum monoccocum and Triticum turgidum gave a negative result, while positive results were obtained in T. aestivum, T. timopheevi, T. zhukovskyi, Secale cereale, Aegilops speltoides, Ae. mutica, Ae. comosa, Ae. caudata, Ae. umbellulata, Ae. squarrosa, and also in the artificial amphiploids (Ae. speltoides x T. monococcum) and (Ae. caudata x T. monococcum).It is concluded that these results agree closely with the classification of Triticum proposed by MacKey in 1966. The investigated protein not only permits the differentiation of T. aestivum from T. turgidum, but also T. turgidum from T. timopheevi at tetraploid level and T. monococcum from all the diploid species of Aegilops.

Protein accumulation, RNA and soluble amino nitrogen content in developing endosperm of two varieties of Triticum aestivum with high and low protein seed.

The kinetics of protein accumulation, the variation in RNA, the soluble amino nitrogen content of developing endosperm of two varieties of Triticum aestivum, with high and low protein content in the mature seed, suggest a possible relation between maintenance of the RNA content and the ability to synthesize protein. A sudden halt in protein accumulation is observed as the RNA starts to decrease. The hypothesis is also advanced that maintenance of the RNA content might, in turn, be dependent on the presence, in the endosperm of developing wheat seed, of a certain level of soluble amino nitrogen which could then play the role of limiting factor for protein synthesis.

[Echography in the study of periskeletal soft tissues: technique, normal and pathologic pictures]. / L'ecografia nello studio dei tessuti molli perischeletrici: tecnica, quadri normali e patologici.

Ultrasonography (US) is the method of choice in the study of normal soft tissue patterns thanks to its intrinsic features. The exam must be performed following strict directions relative to US units (last-generation), to probes (high-frequency, high axial and lateral resolution, focusing), the type of scan (orthogonal and oblique scans), the use of silicon pads. Exam reporting and the exact knowledge of possible image artifacts are of the utmost importance. Normal patterns in the skin, subcutaneous tissue, striate muscles, tendons and bursae, nerves, vessels and bone are reported and correlated with the corresponding US patterns in pathologic conditions. In inflammatory processes, muscles and tendons exhibit blurred, irregular and poorly-defined outlines; neither the anatomical structures nor the tissues nearby are infiltrated. Similar patterns are observed in some benign tumors, while in malignant lesions soft tissue echostructure is more or less alterated and the adjacent tissues are infiltrated. Color-Doppler US yields morphologic and functional information as to pathologic vascularization; typical findings, similar to those obtained by means of angiography, are seen in malignant lesions.

[Echographic assessment of the hand: normal anatomy and pathologic patterns]. / Studio ecografico della mano: anatomia normale e quadri patologici.

Ultrasonography (US) can ben used to study the hand thanks to small-parts probes whose frequency ranges 7.5 MHz to 20 MHz. Due to the complex hand anatomy, the clinician often needs further data relative to periskeletal soft tissues and vessels, which are provided by color Doppler US. Our personal series included 465 patients and a group of 20 healthy volunteers. A silicon pad was used to ensure max. adhesion to probe surface, given the uneven surface of the examined structures. Besides normal anatomic patterns, US demonstrated pathologic features in tendons, sheaths and muscles. As for tendons, stenosing and hypertrophic-exudative tenosynovites were depicted, as well as cysts and ruptures. As for muscles, the main findings were ruptures and, rarely, tumors. Relative to joints, arthrogenous ganglia and rheumatism were observed. Finally, the carpal tunnel syndrome was accurately investigated, where US revealed tenovaginalitis with sheath thickening and deformed and compressed median nerve in the carpal tunnel. Also the benign and malignant masses whose primary location is the nervous tissue are easily demonstrated on US, their typical shape being ovalar. To conclude, US proved extremely accurate in all the surgical patients, exhibiting 100% sensitivity in all of them; its specificity ranged from 84% for tendons, to 81% for muscles, 79% for nervous tissues and finally 82% for joint disorders.

[Role of color coded duplex sonography in the study of thyroid pathology. Personal experience]. / Ruolo della colour coded duplex sonography nello studio della patologia tiroidea. Esperienza personale.

The authors examined 110 patients with suspected pathologic conditions of the thyroid by means of color-coded duplex US. In addition to the information yielded by conventional US, this technique allows organ vascularization to be demonstrated. Five normal patients were considered as a control group: no tissue vascularization was demonstrated in these cases. In 8 patients presenting with carcinoma or recurrences, vascularization was markedly increased both peripheral and central to the nodule. A few doubts are still to be solved as to the diagnostic value of color-coded duplex US in the evaluation of non-carcinomatous nodular pathologic conditions. As a matter of fact, non-functioning avascular adenomas can be demonstrated only in a very low percentage of cases (66%). Color-coded duplex US proved extremely sensitive and specific in depicting malignant neoplasms. Moreover, the use of fine-needle biopsy could be optimized and subsequently reduced. Color-coded duplex US proved to be markedly superior to other methods and techniques in the study of thyroid diseases, especially thyroiditis and multiple pathologic conditions. The simultaneous presence of hypocapture at scintigraphy and peripheral and central vascularization in a single nodule or within multinodular struma at color-Doppler was highly suggestive of malignant thyroid neoplasm. Color-coded duplex US is a low-cost technique, which can be performed on an outpatient basis. Moreover, it is not invasive, nor does it damage the thyroid. That is why its use is almost mandatory in the study of pathologic conditions of the thyroid.

Color Doppler imaging of liver metastases. The value phase-III of a US contrast agent: SH U 508 A (Levovist) Schering.

In a phase-III multicenter clinical trial, the color Doppler vascular patterns were studied of 34 liver metastases in 18 patients before and after the i.v. injection of SH U 508 A (Levovist), with different US units and probes. The patients were selected as having poor color Doppler signals at unenhanced examinations. Lesion size ranged .8 to 9 cm (mean: 3.5 cm). The primary lesion site was the colon in 14 cases, the breast in 8 cases, the lung in 4, the stomach in 4 cases, the ovary in 3 and finally unknown in 4 cases. Each patient received two to four contrast agent injections, with suggested doses and concentrations (10 ml x 300 mg/ml, 8 ml x 400 mgr/ml). No adverse reactions were observed. Thirteen of 34 lesions exhibited no vascular signals at baseline examinations, 10 exhibited some perilesional color spots or small vessel branches, 5 some internal color spots or vessels and 4 small internal and peripheral vessels. After contrast agent infusion, the vascular patterns were better demonstrated in 28/34 lesions and the signal-to-noise ratio was markedly improved, in a concentration-dependent manner, from 40 to 240s. Five lesions remained avascular, 11 exhibited "basket"-like vascular patterns, 10 "internal flow" patterns and finally 10 lesions exhibited both. No major correlation was observed between vascular pattern and lesion size. To conclude, the use of the intravenous contrast agent SH U 508 A (Levovist) appears to be a promising technique to improve the color Doppler demonstration of focal metastatic liver lesions. Nonetheless, further studies on larger series of cases are needed to differentiate the different primary sites of the metastases.

[The role of echotomography in sports traumatology of the lower extremity]. / Ruolo dell'ecotomografia nella traumatologia da sport dell'arto inferiore.

Sonography, which is commonly used in the evaluation of muscular traumas following exercise, quite often provides the radiologist with normal findings. One hundred and fifty-nine athletes were considered (170 US examinations), and the clinical diagnoses the patients had been referred for were compared with US findings: the pathologic condition was confirmed in 59% of the cases, while the extant 41% had normal findings. The highest percentage of positive findings was observed in the Achilles tendon region (75%), while the figure dropped to 38% in the thigh. In the latter location, muscular ruptures were confirmed in 69% of the cases, versus 17% in case of muscular distraction. As for the Achilles tendon, pathologic conditions both along the axis and in the insertion of the tendon were confirmed in 20% of the cases, but clinical accuracy was improved by US in 47% and 20% of the patients, respectively, and the diagnosis was changed in 10% and 20% of the cases, respectively. Therefore, clinical accuracy was demonstrated to depend on the pathologic condition as well as on the anatomical region involved.

[Doppler color in the echographic study of hyperplastic parathyroid glands]. / II contributo del color Doppler nello studio ecografico delle ghiandole paratiroidi iperplasiche.

The sonographic examination of hyperplastic parathyroid glands is a well-known and appreciated technique. However, its diagnostic contribution is still somehow inadequate, due to the difficult differential diagnosis of the various solid hyperechoic nodular structures in the neck and to the presence of frequently ectopic glands. The combined use of B-mode and color-Doppler US allows the vascular features of suspicious parathyroid nodules to be satisfactorily demonstrated. Higher sensitivity and specificity than conventional US are the main advantages of this technique. Still, further research is needed for B-mode color-Doppler US to actually replace fine needle biopsy in confirming the diagnosis.

Serum liver enzymes in Turner syndrome.

UNLABELLED: Increased serum concentrations of liver enzymes are sometimes observed, in the absence of clinical symptoms of liver disease, in patients with Turner syndrome. The purpose of this study was to evaluate, in our Turner patients, serum liver enzyme levels and to find a cause for their increase. In 70 Turner patients, serum AST, ALT, GGT levels were evaluated every 6 months during a period of 0. 8-21.9 years. In patients in whom increased values of liver enzymes were found, serological markers for infectious hepatitis, serum hepatitis C virus RNA and virus genotype, IgG and IgA antibodies to gliadin and endomysium, coeruloplasmin, copper, alpha(1)-antitrypsin, total proteins and electrophoresis, IgG, IgA, IgM, fibrinogen, prothrombin, alkaline phosphatase, creatine kinase and total and direct bilirubin were also determined. Antinuclear, anti-smooth muscle and anti-liver-kidney microsome antibodies together with antithyroglobulin and anti-thyroid peroxidase antibodies were determined in all patients and in 166 age-matched female controls. In 22 patients, increased liver enzymes were observed, not related to karyotype. Follow-up showed that the hepatic disorder did not worsen with the time. Serological markers of hepatitis C virus were positive in three patients. When the serum liver enzyme increase was first observed in the other 19 patients with high enzyme levels (group A), 14 patients had never been submitted to hormonal treatment, 4 were on oestrogen/gestagen treatment and 1 was being treated with both growth hormone and oestrogen. Coeliac disease, alpha(1)-antitrypsin deficiency and Wilson disease were ruled out by appropriate investigations. In 8/19 group A patients, antinuclear and/or anti-smooth muscle antibodies were present versus 6/48 of patients with normal liver enzymes (group B). Thyroid antibodies were found in 8/19 patients in group A and in 13/48 in group B. Weight excess SDS was significantly higher in Turner girls with liver enzyme increase. Ultrasonography, performed in 17 patients of group A, showed mild hepatomegaly in 4 and increased echogenicity with fatty infiltration in 6. CONCLUSION: Hepatic abnormalities in Turner syndrome are not progressive. Oestrogen should not be considered the main cause of increased liver enzymes in Turner syndrome since most of our patients with this finding had not been previously treated with oestrogens. An auto-immune pathogenesis might be considered in some cases, whereas the association with weight excess seems the most frequent cause of liver disorder in Turner syndrome.

[Portal hypertension of hepatic origin. A qualitative assessment by color and echo Doppler US]. / L'ipertensione portale di origine epatica. Valutazione qualitativa mediante color ed eco-Doppler.

One hundred and twenty-eight subjects were studied: 103 of them were affected with portal hypertension diagnosed both radiologically and clinically. Twenty-five healthy subjects were studied, as a control group, by means of combined real-time US and color Doppler. US parameters were evaluated, specific to chronic hepatopathy, together with the Doppler qualitative parameters relative to splanchnic vessels hemodynamics. Our results allowed a sort of noninvasive angiogram of the portal system to be obtained, which is to be of use for diagnosing portal hypertension, and for assessing its causes, risks, and consequences. This study was also aimed at suggesting an examination protocol for portal hypertension, employing real-time and color Doppler US, which any radiologist with enough experience in abdominal US could use. Color Doppler, although not strictly necessary to obtain good results, dramatically shortens execution times. Moreover, color Doppler allows the method to be more quickly learned.

[Color Doppler in the echographic evaluation of breast nodules]. / Il contributo del Color Doppler nella valutazione ecografica dei noduli mammari.

The authors report on their experience with color Doppler sonography in the diagnosis of solid breast masses. Twenty-two patients were examined; breast masses were studied with B-mode US first, and then with color Doppler US to evaluate eventual tumor vascularization. Color Doppler US demonstrated only one vascular pole in histologically confirmed benign masses. On the contrary, in 92.8% of histologically confirmed malignant masses, color Doppler easily depicted 2 or more groups of nutritional arteries. Color Doppler makes the diagnosis of malignant masses easier, thus allowing, in the author's opinion, a reduction in the number of biopsies of solid breast masses clinically/mammographically detected. The use of color Doppler US is therefore suggested: the technique is noninvasive, fast, and easy and its widespread use would translate into advantages for both the patient and the clinician.