[Effects of Heart Rate on Myocardial Native T1 Value Acquired by 5s(3s)3s MOLLI Sequence].

Quantitative evaluation of myocardial native T1 value by measuring modified Look-Locker inversion recovery (MOLLI) method is clinically useful and is used for follow-up of various myocardial diseases. The heart rate during the scan can vary even in the same subjects. Therefore, it is important to know the effects of the heart rate on the native T1 value of the myocardium. In this study, we evaluated the effect of the heart rate on the T1 value in the 5s (3s) 3s scheme, time control data collection period of the MOLLI method, using phantom experiments and experiments of healthy volunteers. The 5s (3s) 3s scheme of the MOLLI method is considered to have little dependence on the heart rate, but the T1 value still varied up to about 7% depending on the heart rate, and was underestimated up to 8% during low heart rate using phantom experiments.

Relationship between Measurement Errors in Myocardial T1 Mapping and Heart Rate.

PURPOSE: Modified Look-Locker inversion recovery (MOLLI) using a 5s(3s)3s scheme is robust to tachycardia, but some errors are occasionally observed in myocardial T1 mapping. We sought to evaluate the relationship between measurement errors in T1 mapping and heart rate (HR) using a confidence map. METHODS: We enrolled 69 male patients with normal native T1 values of the septal myocardium measured by a 5s(3s)3s MOLLI. The degree of measurement errors in the septal myocardium was assessed by two independent observers on a confidence map using a 4-point scale: 0, no errors; 1, errors located on the myocardial contour; 2, errors extended into the myocardial contour; and 3, errors extended into the midwall. We compared the scores of measurement errors and the average, maximum, minimum or variability of the HR indicated during the MOLLI scan (iHR), image phases of MOLLI or left ventricular ejection fraction (LVEF). RESULTS: Patients with score >1 for the septal myocardium had significantly lower minimum iHR than those with a score ≤1 (P < 0.01; 49.8 ± 10.1 vs. 59.6 ± 9.7 beat per min). CONCLUSION: The confidence map shows more measurement errors in patients with lower minimum iHR. The myocardial T1 values should be measured carefully in patients with bradycardia during MOLLI scanning.

PCR-RFLP identification of prohibited animal derived DNA in animal feed.

A method for confirming identification of prohibited species tissue in animal feed has been developed on the basis of PCR-RFLP analysis. In Japan, to prevent the spread of BSE through animal feed, the use of animal protein in feed has been regulated. Species-specific PCR detection of prohibited species materials in feed has been used as one of a series of laboratory tests to ensure the proper implementation of the feed regulations. However, since the result of this PCR method is determined only by amplicon length, it is sometimes necessary to confirm whether or not the positive result is due to the effect of a non-specific reaction. For this purpose, DNA sequencing is the best way to confirm the test result but it is not suitable for routine analysis because of the required time and cost. In this study, we developed an easy and rapid method to confirm the species identification (mammals, ruminants and cattle) by using 4 restriction enzymes: SmlI, MboI, BlnI and Hpy188III. This PCR-RFLP method, which ensures identification of prohibited animal species in feed, is useful for enhancing the reliability of feed inspection for BSE prevention. This method will be added to the Official Methods of Feed Analysis.

Investigation of errors by radiological technologists and evaluation of preventive measures: general and mobile X-ray examinations.

The first objective in this study was to identify the errors of incidents and accidents that occurred in general and mobile X-ray examinations. Based on the analysis of results, the second purpose in this study was to propose useful measures to prevent such errors. As much as 553 radiological technologists in the Gunma Prefecture were surveyed on their experience with errors related to general and mobile X-ray examinations. The questionnaire asked for descriptions of errors experienced during examinations and the responses given (multiple answers possible), and evaluations of the degree of busyness on a five-point scale. A total of 115 questionnaires were returned. Analysis revealed that there was no significant relationship between errors and degree of busyness for either general or mobile examinations. The most frequent error both in general and in mobile examinations was to X-ray a patient mistakenly, the cause of which was cited as failure to confirm the patient's name. After the use of solution priority number to evaluate proposed preventive measures, such as finger-pointing and call, independent double-checks, and verbal self-confirmation would be the simplest and most easily implemented countermeasure.

Impact of potential changes to the current bovine spongiform encephalopathy surveillance programs for slaughter cattle and fallen stock in Japan.

Cattle slaughtered in Japan for human consumption, regardless of their age, have been tested for bovine spongiform encephalopathy (BSE) since October 2001. Beginning in April 2004, all fallen stock from 24 months of age also have been tested. We evaluated the impact of potential changes to the current BSE surveillance programs for both slaughter cattle and fallen stock using a simple stochastic model. We calculated the probability that a BSE-infected dairy cow, Wagyu beef animal, Wagyu-Holstein cross steer or heifer, or Holstein steer slaughtered for human consumption or arising as fallen stock would be tested and detected. Four surveillance strategies were explored for cattle slaughtered for human consumption, with the minimum age at testing set at 0, 21, 31, or 41 months. Three surveillance strategies were explored for fallen stock, with the minimum age at testing set at 24, 31, or 41 months. Increasing the minimum age of testing from 0 to 21 months for both dairy cattle and Wagyu beef cattle had very little impact on the probability that a BSE-infected animal slaughtered for human consumption would be detected. Although increasing the minimum age at testing from 21 to 31 or 41 months would lead to fewer slaughtered animals being tested, the impact on the probability of detecting infected animals would be insignificant. The probability of infected Wagyu-Holstein crosses and Holstein steers being detected at slaughter or as fallen stock would be very low under all surveillance strategies.

Development of primers for detection of heat-treated cetacean materials in porcine meat and bone meal.

The feed ban introduced after the detection of the first case of bovine spongiform encephalopathy in 2001 in Japan has been modified to allow some of the previously prohibited animal materials to be used in animal feed. Recently, porcine materials were allowed to be used in feed for pigs, poultry, and fish. Materials from other mammals, including whales, remain prohibited. In the absence of a method to detect the prohibited whale materials in porcine materials, there is a possibility that the whale materials are being used for feed for pigs, poultry, and fish. To detect illegal use of whale materials mixed with porcine materials, we have developed PCR primers specific to a group of most cetacean species, using a computer-based method we developed previously. The primer sets were capable of detecting whale meat meal that had been autoclaved at 133 degrees C for up to 20 min. The detection limit of whale material in porcine meat and bone meal was 0.1%.

Development of PCR primers for the detection of porcine DNA in feed using mtATP6 as the target sequence.

In Japan, PCR identification of species-specific, animal group-specific and plant DNA is employed as part of the audit program to ensure compliance with the feed ban in place for the control of bovine spongiform encephalopathy (BSE). Since October 2001, animal proteins other than dairy proteins, egg proteins and gelatin have been prohibited to be used in feed for ruminants. Meat-and-bone meal (MBM) derived from poultry, pig and/or fish is allowed to be used in feed for poultry, pigs and fish. Porcine MBM is permitted in feed for domestic animals other than cattle since April 2005. Given the fact that pigs and cattle are the two major sources of MBM in Japan, the identification of porcine DNA with high specificity and sensitivity has become increasingly important to ensure that MBM products are free from ruminant materials. Two PCR primer sets (PPA8 and PPA6) were newly designed using mtATP8 and mtATP6 as the target sequences, with relatively short amplification sizes. PPA8 and PPA6 were able to specifically detect porcine DNA with the detection limits of 0.01% and 0.001% of porcine MBM in feed, respectively. PPA6 was superior to PPA8 in terms of detection of DNA damaged/fragmented during rendering procedures. The PCR method using these primer sets is registered as the official analytical method for feed in Japan.

Risk of introduction of BSE into Japan by the historical importation of live cattle from the United Kingdom.

All cattle imported from the United Kingdom to Japan since 1980 and slaughtered before 2002 were traced (n=33), and the number of cattle that were possibly infected with BSE and entered the animal feed chain was calculated. Because there was no effective system to avoid recycling of the BSE agent via animal feed until the early 1990s, of the 33 cattle imported from the UK into Japan, most probably 7 or 8 were infected and entered the animal feed chain, 2 of which entered the animal feed chain in each of 1992 and 1993. In terms of infectivity, 400-550 cattle oral ID(50) of the BSE agent entered the feed chain in each of these years. The amount of infectivity that entered the feed chain in 1989, 1991 and 1995 was smaller but still substantial, suggesting that the BSE agent might have entered the Japanese feed chain in any of these years.

High GC contents of primer 5'-end increases reaction efficiency in polymerase chain reaction.

Many studies have suggested that regulation of the polymerase chain reaction (PCR) is influenced by several factors. However, the understanding of reaction efficiency factors is not sufficient. Here we propose that high GC contents of primer 5'-end increases reaction efficiency in PCR. Using 71 primers (45 pairs), we analyzed factors that affect reaction efficiency, and statistically tested the correlation between the amplification signals and several factors. As a result, there were significant correlations between the amplification signals and the GC contents in the first 1 approximately 3 bps of primer 5'-end.

Developing PCR primers using a new computer program for detection of multiple animal-derived materials in feed.

To reduce the risk of the bovine spongiform encephalopathy agent being recycled to cattle through animal feed, in October 2001 Japan introduced a feed ban prohibiting the use of animal proteins in feed. PCR identification of feed ingredients is part of the audit program to ensure the proper implementation of the feed ban. For efficient analysis, screening of feed products for materials from multiple species is essential. In our study, we developed a computer program GSPRIMER (http://www. famic.go.jp/ffis/feed/gsprimer/) that facilitates development of PCR primers specific to multiple species. The most important feature of GSPRIMER is its ability to estimate the specificity and homology of a potential primer in incremental steps from the 3' terminal. We analyzed all regions of mitochondrial DNA from the target and nontarget species using GSPRIMER. We designed species-specific primer sets for three animal species (sheep, goats, and swine) and group-specific primer sets for ruminants and animals susceptible to transmissible spongiform encephalopathy. The primers were efficiently screened by the PCR protocol using a mixture of mitochondrial DNA from nontarget species as a template. As a result, one primer set each for sheep and goats, two for swine, and three for a group of ruminant species were developed. The detection limit of one of the ruminant primer sets ranged from 0.05 to 0.01% bovine meat and bone meal and 0.1 pg of bovine DNA. We also successfully applied the primer set to 17 commercial feed samples that were known to be free from ruminant-derived materials. No false-positive results were found.

Interlaboratory validation of the modified murine local lymph node assay based on adenosine triphosphate measurement.

INTRODUCTION: The murine local lymph node assay (LLNA) is a well-established alternative to the guinea pig maximization test (GPMT) or Buehler test (BT) for the assessment of the skin sensitizing ability of drugs and chemicals. Daicel Chemical Industries Ltd. has developed a modified LLNA based on the adenosine triphosphate (ATP) content (LLNA-DA). We conducted 2 interlaboratory validation studies to evaluate the reliability and relevance of LLNA-DA. METHODS: The experiment involved 17 laboratories, wherein 14 chemicals were examined under blinded conditions. In the first study, 3 chemicals were examined in 10 laboratories and the remaining 9 were examined in 3 laboratories. In the second study, 1 chemical was examined in 7 laboratories and the remaining 4 chemicals were examined in 4 laboratories. The data were expressed as the ATP content for each chemical-treated group, and the stimulation index (SI) for each chemical-treated group was determined as the increase in the ATP content relative to the concurrent vehicle control group. An SI of 3 was set as the cut-off value for exhibiting skin sensitization activity. RESULTS: The results of the first study obtained in the experiments conducted for the 3 chemicals that were examined in all the 10 laboratories and for 5 of the remaining 9 chemicals were sufficiently consistent with small variations in their SI values. The sensitivity, specificity, and accuracy of LLNA-DA against those of GPMT/BT were 7/8 (87.5%), 3/3 (100%), and 10/11 (90.9%), respectively. In the second study, all the 5 chemicals studied demonstrated acceptably small interlaboratory variations. DISCUSSION: In the first study, a large variation was observed for 2 chemicals; in the second study, this variation was small. It was attributed to the application of dimethylsulfoxide as the solvent for the metallic salts. In conclusion, these 2 studies provide good evidence for the reliability of the LLNA-DA.