Assessment of ejection fraction and heart perfusion using myocardial perfusion single-photon emission computed tomography in Finland and Estonia: a multicenter phantom study.

OBJECTIVES: Myocardial SPECT/CT imaging is frequently performed to assess myocardial perfusion and dynamic parameters of heart function, such as ejection fraction (EF). However, potential pitfalls exist in the imaging chain that can unfavorably affect diagnosis and treatment. We performed a national cardiac quality control study to investigate how much SPECT/CT protocols vary between different nuclear medicine units in Finland, and how this may affect the heart perfusion and EF values. METHODS: Altogether, 21 nuclear medicine units participated with 27 traditional SPECT/CT systems and two cardiac-centered IQ-SPECT systems. The reproducibility of EF and the uniformity of perfusion were studied using a commercial dynamic heart phantom. SPECT/CT acquisitions were performed and processed at each participating unit using their own clinical protocol and with a standardized protocol. The effects of acquisition protocols and analysis routines on EF estimates and uniformity of perfusion were studied. RESULTS: Considerable variation in EF estimates and in the uniformity of perfusion were observed between the units. Uniformity of perfusion was improved in some units after applying the higher count-statistic standard acquisition protocol. EF estimates varied more due to differences in analysis routines than as a result of different acquisition protocols. The results obtained with the two IQ-SPECT systems differed substantially from the traditional multipurpose cameras. CONCLUSION: On average, the EF and heart perfusion were accurately estimated by SPECT/CT, but high errors could be produced if the acquisition and analysis routines were poorly optimized. Eight of the 21 participants altered their imaging protocol after this quality control tour.

Multicenter evaluation of renography with an automated physical phantom.

PURPOSE: The diversity of the dynamic radionuclide renal imaging (renography) study protocols sets challenges for the overall study quality, therefore raising a need for national quality control. The aim of this study was to encourage the standardization of renography in Finland and to evaluate the development after a previous study performed in 1997. METHODS: The new Heikkinen phantom was imaged in each of the 20 participating nuclear medicine laboratories. The results were interpreted in the manner of a regular patient study, and reconstructions and printouts were made according to the clinical routines of each laboratory. Four quantitative parameters were calculated and compared between laboratories. The reports were also assessed in a blind test. RESULTS: The average error in T(max) values ranged from -5 to 7% (-29 to +18% in 1997), in T(1/2) from 0 to 35% (-43 to +66%), in RCA20 from -20 to +28% (-50 to +82%) and in relative uptake from -3 to 5%. The difference from average in relative uptake ranged from -4 to 5% (-21 to +36%). CONCLUSION: The results showed that the errors in T(max) and relative uptake were generally within quite acceptable margins, and the variation in quantitative parameters between laboratories was shown to be smaller than 14 years earlier. The reason might be the use of new software packages as well as increased efforts to improve the quality of the studies.

UK audit of analysis of quantitative parameters from renography data generated using a physical phantom.

BACKGROUND: In this second UK audit of quantitative parameters obtained from renography, phantom simulations were used in cases in which the 'true' values could be estimated, allowing the accuracy of the parameters measured to be assessed. MATERIALS AND METHODS: A renal physical phantom was used to generate a set of three phantom simulations (six kidney functions) acquired on three different gamma camera systems. A total of nine phantom simulations and three real patient studies were distributed to UK hospitals participating in the audit. Centres were asked to provide results for the following parameters: relative function and time-to-peak (whole kidney and cortical region). As with previous audits, a questionnaire collated information on methodology. Errors were assessed as the root mean square deviation from the true value. RESULTS: Sixty-one centres responded to the audit, with some hospitals providing multiple sets of results. Twenty-one centres provided a complete set of parameter measurements. Relative function and time-to-peak showed a reasonable degree of accuracy and precision in most UK centres. The overall average root mean squared deviation of the results for (i) the time-to-peak measurement for the whole kidney and (ii) the relative function measurement from the true value was 7.7 and 4.5%, respectively. These results showed a measure of consistency in the relative function and time-to-peak that was similar to the results reported in a previous renogram audit by our group. CONCLUSION: Analysis of audit data suggests a reasonable degree of accuracy in the quantification of renography function using relative function and time-to-peak measurements. However, it is reasonable to conclude that the objectives of the audit could not be fully realized because of the limitations of the mechanical phantom in providing true values for renal parameters.

Dimerization of human lysyl hydroxylase 3 (LH3) is mediated by the amino acids 541-547.

Lysyl hydroxylases (LH), which catalyze the post-translational modifications of lysines in collagen and collagen-like proteins, function as dimers. However, the amino acids responsible for dimerization and the role of dimer formation in the enzymatic activities of LH have not yet been identified. We have localized the region responsible for the dimerization of lysyl hydroxylase 3 (LH3), a multifunctional enzyme of collagen biosynthesis, to a sequence of amino acids between the glycosyltransferase activity and the lysyl hydroxylase activity domains. This area is covered by amino acids 541-547 in human LH3, but contains no cysteine residues. The region is highly conserved among LH isoforms, and is also involved in the dimerization of LH1 subunits. Dimerization is required for the LH activity of LH3, whereas it is not obligatory for the glycosyltransferase activities. In order to determine whether complex formation can occur between LH molecules originating from different species, and between different LH isoforms, double expressions were generated in a baculovirus system. Heterocomplex formation between mouse and human LH3, between human LH1 and LH3 and between human LH2 and LH3 was detected by western blot analyses. However, due to the low amount of complexes formed, the in vivo function of heterocomplexes remains unclear.

Ability of ultrasound imaging to detect erosions in a bone phantom model.

OBJECTIVES: The authors examined the validity, interobserver reliability and interscanner variation in detecting bone erosions with ultrasonography using a custom-made phantom. METHODS: 21 bovine bones were used. Artificial erosions were made into 15 bones and six bones were left as controls. In the processed bones the numbers of erosions, their depths and widths varied between 1-7, 1-4 and 1.5-5 mm, respectively. Each bone was coated with polyvinyl alcohol cryogel to mimic overlying soft tissue and to hide the erosions. Four musculoskeletal sonography experts scanned the 21 blind-coded phantoms using one of the three sets of ultrasound equipment. Finally, quality assurance measurements of the ultrasound equipment was carried out using two additional bone samples. RESULTS: The sonographers detected the erosions successfully with ultrasound. The mean correlation coefficient for a correct result in terms of the number of erosions detected was 0.88 (range 0.75-0.975). The overall Cohen's kappa coefficient for interobserver agreement was 0.683 in terms of discrimination between healthy bones and bones with erosions. The different sets of equipment showed that their overall performance was equal. CONCLUSIONS: The sonographers had good correlations with the number of erosions and they were successful in separating healthy bones from bones with erosions. It seems that neither depth nor width is crucial but that in experimental conditions a 1.5 mm erosion width was the limit for the resolution with current ultrasound equipment. Ultrasound is a valid and reliable method of detecting cortical bone erosions in vitro, when the round erosion is at least 1 mm deep and 1.5 mm wide.

Influence of 12 weeks of jogging on magnetic resonance-determined left ventricular characteristics in previously sedentary subjects free of cardiovascular disease.

Hypertrophy of the left ventricle is a diagnostic dilemma in subjects who engage in regular endurance exercise. We studied prospectively whether endurance training in previously sedentary young and middle-aged men and women can alter left ventricular (LV) characteristics. We recruited 33 healthy young and middle-aged subjects (18 women, 15 men, ages 21 to 59 years) to undergo 12 weeks of home-based brisk walking and jogging at a target heart rate > or =120 beats/min for > or =30 minutes 3 times a week. LV characteristics were measured by cine magnetic resonance imaging. Training intensity as estimated by heart rate correlated positively with the increase in LV myocardial area (r = 0.51, p = 0.005) in the 28 men and women completing the study. In the 13 men and women who trained with heart rate of > or =120 beats/min, LV myocardial area was larger after than before training (17.7 +/- 2.9 vs 16.8 +/- 2.8 cm(2), p <0.05). Moreover, in these subjects LV myocardial area increased more (5.5 +/- 9.0% vs -3.0 +/- 5.0%) than in the 15 men and women who trained at a lower intensity (p <0.05). LV end-systolic and end-diastolic area and ejection fraction did not change significantly. In conclusion, moderate-to-vigorous endurance training at moderate volumes does not influence LV end-diastolic volume or ejection fraction, but has a minor influence on LV hypertrophy in previously sedentary young and middle-aged men and women.

Role of quantitative cholescintigraphy for planning laparoscopic cholecystectomy in patients with gallbladder dyskinesia and chronic abdominal pain.

Our aim was to examine the diagnostic role and therapeutic guide of quantitative cholescintigraphy in 122 patients with a biliary-type chronic abdominal pain and normal abdominal ultrasound. The patients with severe symptoms and an impaired ejection fraction (EF35%, n=38) EF. An impaired gallbladder emptying was found in 84/122 of patients (69%) and LC was performed in 32/122 (26%) of the patients, that is, 2.2% of all cholecystectomies in our hospital. After the mean follow-up of 4 years, chronic abdominal pain was totally improved or diminished in 92% in the LC group, 45% in the nonoperative group with EF35%, respectively (P=0.0003). Quantitative cholescintigraphy is a useful aid when considering the treatment options in patients with chronic biliary-type abdominal pain and a negative abdominal ultrasound.

Expanding the lysyl hydroxylase toolbox: new insights into the localization and activities of lysyl hydroxylase 3 (LH3).

Hydroxylysine and its glycosylated forms, galactosylhydroxylysine and glucosylgalactosylhydroxylysine, are post-translational modifications unique to collagenous sequences. They are found in collagens and in many proteins having a collagenous domain in their structure. Since the last published reviews, significant new data have accumulated regarding these modifications. One of the lysyl hydroxylase isoforms, lysyl hydroxylase 3 (LH3), has been shown to possess three catalytic activities required sequentially to produce hydroxylysine and its glycosylated forms, that is, the lysyl hydroxylase (LH), galactosyltransferase (GT), and glucosyltransferase (GGT) activities. Studies on mouse models have revealed the importance of these different activities of LH3 in vivo. LH3 is the main molecule responsible for GGT activity in mouse embryos. A lack of this activity causes intracellular accumulation of type IV collagen, which disrupts the formation of basement membranes (BMs) during mouse embryogenesis and leads to embryonic lethality. The specific inactivation of the LH activity of LH3 causes minor alterations in the structure of the BM and collagen fibril organization, but does not affect the lifespan of mutated mice. Recent data from zebrafish demonstrate that growth cone migration depends critically on the LH3 glycosyltransferase domain. LH3 is located in the ER loosely associated with the membranes, but, unlike the other isoforms, LH3 is also found in the extracellular space in some tissues. LH3 is able to adjust the amount of hydroxylysine and hydroxylysine-linked carbohydrates of extracellular proteins in their native conformation, suggesting that it may have a role in matrix remodeling.

Interdepartmental audit with an anatomically realistic lung phantom.

UNLABELLED: The diagnostic proficiency of nuclear medicine professionals and the accuracy of equipment may be tested with phantoms. All phases of the imaging chain should be included in the external quality assurance of imaging. METHODS: The aim of this study was to evaluate and compare the quality of nuclear imaging of the lung in Finland. For this purpose, we developed a new anatomically realistic lung phantom. The phantom consisted of plastic containers filled with plastic pellets to imitate the 3-dimensional shape of the lungs. These containers were filled with radioactive liquid and placed inside an anatomically accurate phantom of the chest cavity. The attenuation properties of the phantom were close to those of a real human thorax. Perfusion and ventilation defects were positioned inside the phantom to mimic 2 clinical cases. The phantom was imaged and interpreted as a patient simulation study in 18 Finnish hospitals. Reconstruction, printout, and reporting were according to the clinical routine of each hospital. The quality of the image sets and reports was evaluated and scored from 0 to 10. Additionally, technical performance was evaluated by a nuclear medicine specialist and hospital physicians. RESULTS: The average score (+/-SD) for overall quality was 7.1+/-1.1 (range, 5.2-8.5). Reports received a score of 7.2+/-1.7 (4.7-10.0); image sets, 7.2+/-1.3 (4.8-9.7), technical evaluation by hospital readers, 6.5+/-2.3 (1.6-9.5); and technical evaluation by a specialist, 7.8+/-1.2 (5.7-10.0). CONCLUSION: Lung imaging routines and the results of this survey were diverse. None of the participating hospitals routinely used tomography. In planar imaging, the most valuable projections were oblique (left anterior oblique, right anterior oblique, left posterior oblique, and right posterior oblique) and straight sides (right and left). The phantom mimics variable clinical situations well and is suitable for testing of imaging protocols and for proficiency testing of nuclear medicine professionals and equipment. Clinical phantom studies are an effective way of assessing an imaging program.

Lysyl hydroxylase 3 (LH3) modifies proteins in the extracellular space, a novel mechanism for matrix remodeling.

Lysyl hydroxylase 3 (LH3), the multifunctional enzyme associated with collagen biosynthesis that possesses lysyl hydroxylase and collagen glycosyltransferase activities, has been characterized in the extracellular space in this study. Lysine modifications are known to occur in the endoplasmic reticulum (ER) prior to collagen triple-helix formation, but in this study we show that LH3 is also present and active in the extracellular space. Studies with in vitro cultured cells indicate that LH3, in addition to being an ER resident, is secreted from the cells and is found both in the medium and on the cell surface associated with collagens or other proteins with collagenous sequences. Furthermore, in vivo, LH3 is present in serum. LH3 protein levels correlate with the galactosylhydroxylysine glucosyltransferase (GGT) activity of mouse tissues. This, together with other data, indicates that LH3 is responsible for GGT activity in the tissues and that GGT activity assays can be used to quantify LH3 in tissues. LH3 in vivo is located in two compartments, in the ER and in the extracellular space, and the partitioning varies with tissue type. In mouse kidney the enzyme is located mainly intracellularly, whereas in mouse liver it is located solely in the extracellular space. The extracellular localization and the ability of LH3 to modify lysyl residues of extracellular proteins in their native, nondenaturated conformation reveals a new dynamic in extracellular matrix remodeling, suggesting a novel mechanism for adjusting the amount of hydroxylysine and hydroxylysine-linked carbohydrates in collagenous proteins.

Localization of sentinel nodes in breast cancer: novel method and device to help pen marking of active nodes during gamma camera imaging.

Gamma camera imaging with Tc-99m marking is a widely used method to locate sentinel lymph nodes (SNs) in breast cancer patients. Prior to SN biopsy, the anterior and lateral location of the SN is marked on the patient's skin using an ink pen. The pen marks guide the surgeon during an operation. However, in many cases the marking is difficult due to limited space under the detectors of a gamma camera. The aim of this study was to improve the pen marking method. Eleven female patients were imaged 3-4 h after injection of Tc-99m labelled Nanocol. Injection was performed to parenchyma surrounding the breast tumour. To facilitate pen marking, two polycarbonate (PC) plates with 40 x 32 holes (spacing=10 mm) were engineered for anterior and lateral side imaging and then installed on the bed of a dual-head gamma camera. Two drops of Tc-99m were placed into the top corners of both the PC plates, in order to trace the corresponding x-y coordinates first from the acquired images and then from the plates. After imaging, the x-y coordinates of the SN(s) were determined from the anterior and lateral side images. Subsequently, the location of each SN was marked with an ink pen on the skin through the small holes in the PC plates. According to the surgeon's evaluation, the distance between the marks and the true location of the SNs was 4.5+/-6.9 mm. Measurements with a custom made phantom revealed that the accuracy of the novel method was significantly (P=0.06) higher as compared with the traditional method (2.7+/-3.0 mm versus 9.2+/-3.0 mm). In addition, we were not able to mark the weakest activity (0.02 MBq) with the traditional method. Taken together, the marking process was considerably easier with the novel method, it had better accuracy and sensitivity than the traditional method and the device is simple enough to be adapted for most gamma cameras.

New automated physical phantom for renography.

UNLABELLED: Physical phantoms have been used to test the diagnostic proficiency of nuclear medicine professionals and the accuracy of their equipment in external quality assurance surveys. No dynamic renal phantoms are commercially available. A new renal phantom, presented in this paper, was constructed and patented in the United States. METHODS: The organs to be simulated by the phantom were in the form of containers filled with radioactive solution, and the device further comprised movable steel and lead plates between the containers and the gamma-camera. The detectable radiation was regulated in accordance with automated computer-controlled step motors to move the attenuators to simulate a given patient situation. The reproducibility of the phantom measurements was defined as a coefficient of variation. Four different kidney-function simulations were repeated 3 times, and 6 parameters were compared. RESULTS: The average root mean square deviation of the coefficient of variation was 6.7% for the perfusion integral, 1.3% for time to reach the maximum activity, 19.7% for mean transit time, 3.3% for function (Patlak [%]), 1.0% for outflow index (%), and 6.5% for time to reach the half-activity from maximum. CONCLUSION: With this phantom, the true values of most parameters measured are well known; it closely approaches true extraction, washout, and attenuation properties and curves, and the images produced are similar to those of patient studies. Compared with the first manual version, this new automated phantom is easy to use. Any desired clinical situation can be programmed. It is a promising tool for quality assurance and calibration of renography.

The third activity for lysyl hydroxylase 3: galactosylation of hydroxylysyl residues in collagens in vitro.

Lysyl hydroxylase (LH, EC 1.14.11.4), galactosyltransferase (EC 2.4.1.50) and glucosyltransferase (EC 2.4.1.66) are enzymes involved in posttranslational modifications of collagens. They sequentially modify lysyl residues in specific positions to hydroxylysyl, galactosylhydroxylysyl and glucosylgalactosyl hydroxylysyl residues. These structures are unique to collagens and essential for their functional activity. Lysines and hydroxylysines form collagen cross-links. Hydroxylysine derived cross-links, usually as glycosylated forms, occur especially in weight-bearing and mineralized tissues. The detailed functions of the hydroxylysyl and hydroxylysyl linked carbohydrate structures are not known, however. Hydroxylysine linked carbohydrates are found mainly in collagens, but recent reports indicate that these structures are also present and probably have an important function in other proteins. Earlier we have shown that human LH3, but not isoforms LH1, LH2a and LH2b, possesses both LH and glucosyltransferase activity (J. Biol. Chem. 275 (2000) 36158). In this paper we demonstrate that galactosyltransferase activity is also associated with the same gene product, thus indicating that one gene product can catalyze all three consecutive steps in hydroxylysine linked carbohydrate formation. In vitro mutagenesis experiments indicate that Cys(144) and aspartates in positions 187-191 of LH3 are important for the galactosyltransferase activity. Our results suggest that manipulation of the gene for LH3 can be used to selectively alter the glycosylation and hydroxylation reactions, and provides a new tool to clarify the functions of the unique hydroxylysine linked carbohydrates in collagens and other proteins.

Identification of amino acids important for the catalytic activity of the collagen glucosyltransferase associated with the multifunctional lysyl hydroxylase 3 (LH3).

Collagen glucosyltransferase (GGT) activity has recently been shown to be associated with human lysyl hydroxylase (LH) isoform 3 (LH3) (Heikkinen, J., Risteli, M., Wang, C., Latvala, J., Rossi, M., Valtavaara, M., Myllylä, R. (2000) J. Biol. Chem. 275, 36158-36163). The LH and GGT activities of the multifunctional LH3 protein modify lysyl residues in collagens posttranslationally to form hydroxylysyl and glucosylgalactosyl hydroxylysyl residues respectively. We now report that in the nematode, Caenorhabditis elegans, where only one ortholog is found for lysyl hydroxylase, the LH and GGT activities are also associated with the same gene product. The aim of the present studies is the identification of amino acids important for the catalytic activity of GGT. Our data indicate that the GGT active site is separate from the carboxyl-terminal LH active site of human LH3, the amino acids important for the GGT activity being located at the amino-terminal part of the molecule. Site-directed mutagenesis of a conserved cysteine at position 144 to isoleucine and a leucine at position 208 to isoleucine caused a marked reduction in GGT activity. These amino acids were conserved in C. elegans LH and mammalian LH3, but not in LH1 or LH2, which lack GGT activity. The data also reveal a DXD-like motif in LH3 characteristic of many glycosyltransferases and the mutagenesis of aspartates of this motif eliminated the GGT activity. Reduction in GGT activity was not accompanied by a change in the LH activity of the molecule. Thus GGT activity can be manipulated independently of LH activity in LH3. These data provide the information needed to design knock-out studies for investigation of the function of glucosylgalactosyl hydroxylysyl residues of collagens in vivo.