[Report of immunoassay control surveys conducted in the past 30 years by the Subcommittee for Radioisotope in vitro Test, the Medical Science and Pharmaceutical Committee, and the Japan Radioisotope Association].

Immunoassay control surveys, were conducted by the Subcommittee for Radioisotope in vitro Test, the Medical Science and Pharmaceutical Committee, and the Japan Radioisotope Association, between 1978 to 2008. A total of 40 analytes for 26 hormones, 14 tumor markers and pharmaceutical drugs were investigated in participating facilities. In the first immunoassay control survey in 1978, samples were measured using only RI kits, however, non-RI kits increased gradually during the next 30 years. In the 30th immunoassay control survey, more than 90% samples were measured using non-RI kits. Coefficient variation (CV) of intra-kits has been decreasing yearly in all analytes for hormones as well as tumor markers. However, improvement of CV in inter-kits has not been seen in the past 30 years by a lack of international standards, although there has been continuous effort over the years for the standardization of immunoassay. Growth hormone (GH) deficiency has been diagnosed using various loading tests. However, the clinical diagnosis varies according to the GH kit used. Standardization for GH measurement has been possible by using recombinant GH as the standard among commercial GH kits. The diagnosis of subclinical Cushing's syndrome also varies according to the cortisol kits being used. Candidate reference measurement procedure and low level cortisol standards have been developed by the Biomedical Standard Section, of the National Metrology Institute of Japan. Standardization of measurement is necessary for improvement of immunoassay.

[Thyroid function tests and thyroid autoantibody tests, a review].

Recent progress and present situation of the thyroid function tests and the thyroid autoantibody tests were reviewed with some author's opinion. Recent clinical laboratory examinations using immunoassay are mainly non-isotopic full-automatic assay with various kinds of methodological principal. These bring us difficulties of the standardization of the tests. On the other hand, recent immunoassay enables us extremely quick turn-around-time in the clinical practice. Pre-consultation examination is widely carried out in our county. Clinical guidelines for many thyroid diseases are discussed and distributed recently. We have to pay very careful attention to the clinical examination results from non-standardized immunoassay. We need enough comprehension of the principal differences among tests used in the clinical practice.

Report of the IFCC Working Group for Standardization of Thyroid Function Tests; part 3: total thyroxine and total triiodothyronine.

BACKGROUND: Because total thyroid hormone testing is performed on many automated clinical chemistry instruments, the IFCC Scientific Division commissioned the Working Group for Standardization of Thyroid Function Tests to include total thyroxine (TT4) and total triiodothyronine (TT3) in its standardization efforts. METHODS: Existing SI-traceable reference measurement procedures (RMPs) were used to assign TT4 and TT3 values to 40 single-donor serum samples for subsequent use in a method comparison study with 11 TT4 and 12 TT3 immunoassays. Data from comparison of each immunoassay with the RMPs provided a basis for mathematical assay recalibration. RESULTS: Seven TT4 assays had a mean bias within 10% of the RMP, but 2 deviated by an average of -12% and another 2 by +17%. All TT3 assays showed positive biases, 4 within and 8 outside 10%, up to 32%. Mathematical recalibration effectively eliminated assay-specific biases, but sample-related effects remained, particularly for TT3. Correlation coefficients with the RMPs ranged from 0.82 to 0.97 for TT4 and from 0.32 to 0.92 for TT3. The within-run and total imprecision ranges for TT4 were 1.4% to 9.1% and 3.0% to 9.4%, respectively, and for TT3 2.1% to 7.8% and 2.8% to 12.7%, respectively. Approximately one-half of the assays matched the internal QC targets within approximately 5%; however, we observed within-run drifts/shifts. CONCLUSIONS: The study showed that of the assays we examined, only 4 TT4 but the majority of the TT3 assays needed establishment of calibration traceability to the existing RMPs. Most assays performed well, but some would benefit from improved precision, within-run stability, and between-run consistency.

Report of the IFCC Working Group for Standardization of Thyroid Function Tests; part 2: free thyroxine and free triiodothyronine.

BACKGROUND: Free thyroxine (FT4) and free triiodothyronine (FT3) measurements are useful in the diagnosis and treatment of a variety of thyroid disorders. The IFCC Scientific Division established a Working Group to resolve issues of method performance to meet clinical requirements. METHODS: We compared results for measurement of a panel of single donor sera using clinical laboratory procedures based on equilibrium dialysis-isotope dilution-mass spectrometry (ED-ID-MS) (2 for FT4, 1 for FT3) and immunoassays from 9 manufacturers (15 for FT4, 13 for FT3) to a candidate international conventional reference measurement procedure (cRMP) also based on ED-ID-MS. RESULTS: For FT4 (FT3), the mean bias of 2 (4) assays was within 10% of the cRMP, whereas for 15 (9) assays, negative biases up to -42% (-30%) were seen; 1 FT3 assay was positively biased by +22%. Recalibration to the cRMP eliminated assay-specific biases; however, sample-related effects remained, as judged from difference plots with biologic total error limits. Correlation coefficients to the cRMPs ranged for FT4 (FT3) from 0.92 to 0.78 (0.88 to 0.30). Within-run and total imprecision ranged for FT4 (FT3) from 1.0% to 11.1% (1.8% to 9.4%) and 1.5% to 14.1% (2.4% to 10.0%), respectively. Approximately half of the manufacturers matched the internal QC targets within approximately 5%; however, within-run instability was observed. CONCLUSIONS: The study showed that most assays had bias largely correctable by establishing calibration traceability to a cRMP and that the majority performed well. Some assays, however, would benefit from improved precision, within-run stability, and between-run consistency.

Report of the IFCC Working Group for Standardization of Thyroid Function Tests; part 1: thyroid-stimulating hormone.

BACKGROUND: Laboratory testing of serum thyroid-stimulating hormone (TSH) is an essential tool for the diagnosis and management of various thyroid disorders whose collective prevalence lies between 4% and 8%. However, between-assay discrepancies in TSH results limit the application of clinical practice guidelines. METHODS: We performed a method comparison study with 40 sera to assess the result comparability and performance attributes of 16 immunoassays. RESULTS: Thirteen of 16 assays gave mean results within 10% of the overall mean. The difference between the most extreme means was 39%. Assay-specific biases could be eliminated by recalibration to the overall mean. After recalibration of singlicate results, all assays showed results within the biological total error goal (22.8%), except for 1 result in each of 4 assays. For a sample with a TSH concentration of 0.016 mIU/L, 6 assays either did not report results or demonstrated CVs >20%. Within-run and total imprecision ranged from 1.5% to 5.5% and 2.5% to 7.7%, respectively. Most assays were able to match the internal QC targets within 5%. Within-run drifts and shifts were observed. CONCLUSIONS: Harmonization of TSH measurements would be particularly beneficial for 3 of the 16 examined assays. These data demonstrate that harmonization may be accomplished by establishing calibration traceability to the overall mean values for a panel of patient samples. However, the full impact of the approach must be further explored with a wider range of samples. Although a majority of assays showed excellent quality of performance, some would benefit from improved within-run stability.

[In vitro susceptibilities to levofloxacin and various antibacterial agents of 12,919 clinical isolates obtained from 72 centers in 2007].

We have reported in this journal in vitro susceptibilities of clinical isolates to antibiotics every year since 1992. In this paper, we report the results of an analysis of in vitro susceptibilities of 12,919 clinical isolates from 72 centers in Japan to selected antibiotics in 2007 compared with the results from previous years. The common respiratory pathogens, Streptococcus pyogenes, Streptococcus pneumoniae, Moraxella catarrhalis and Haemophilus influenzae maintained a high susceptibility to fluoroquinolones (FQs). The resistance of S. pyogenes to macrolides has been increasing every year and this was especially clear this year. Most strains of Enterobacteriaceae except for Escherichia coli showed a high susceptibility to FQs. Almost 30% of E. coli strains were resistant to FQs and the resistance increased further this year. FQs resistance of methicillin-resistant Staphylococcus aureus (MRSA) was approximately 95% with the exception of 45% for sitafloxacin (STFX). FQs resistance of methicillin-susceptible S. aureus (MSSA) was low at about 10%. FQs resistance of methicillin-resistant coagulase negative Staphylococci (MRCNS) was higher than that of methicillin-susceptible coagulase negative Staphylococci (MSCNS), but it was lower than that of MRSA. However, FQs resistance of MSCNS was higher than that of MSSA. FQs resistance of Enterococcus faecalis was 22.5% to 29.6%, while that of Enterococcusfaecium was more than 85% except for STFX (58.3%). In clinical isolates of Pseudomonas aeruginosa derived from urinary tract infections, FQs resistance was 21-27%, which was higher than that of P. aeruginosa from respiratory tract infections at 13-21%, which was the same trend as in past years. Multidrug resistant strains accounted for 5.6% in the urinary tract and 1.8% in the respiratory tract. Acinetobacter spp. showed high susceptibility to FQs. The carbapenem resistant strains, which present a problem at present, accounted for 2.7%. Neisseria gonorrhoeae showed high resistance of 86-88% to FQs. The results of the present survey indicated that although methicillin-resistant Staphylococci, Enterococci, E. coli, P. aeruginosa, and N. gonorrhoeae showed resistance tendencies, and other species maintained high susceptibility rates more than 90% against FQs, which have been used clinically for over 15 years.

A novel homozygous missense mutation of the dual oxidase 2 (DUOX2) gene in an adult patient with large goiter.

OBJECTIVE: To describe the first adult case of large goiter associated with a novel R1110Q mutation in the dual oxidase 2 (DUOX2) gene. She was initially euthyroid, and developed hypothyroidism later in her forties. DUOX2 is an essential enzyme in iodine organification of thyroid hormone biosynthesis. Only infant cases of congenital hypothyroidism due to mutations of the DUOX2 gene have been reported. Biallelic mutation of DUOX2 is thought to lead to total iodine organification defect. PATIENTS AND MEASUREMENT: This 57-year-old woman became first aware of goiter around the age of 20 years. Since the goiter had enlarged gradually, she consulted us at the age of 32 years. Goiter was soft, and thyroid function was normal. Antithyroid antibodies were negative. Both physical and mental development was normal. Three of her nine siblings and her mother had large goiters. At the age of 44 years, thyroid function demonstrated subclinical hypothyroidism. She started to take levo-thyroxine at a dose of 100 mug/day to reduce goiter. At the age of 56 years, goiter size remained the same. The perchlorate discharge rate was 72.8%, suggesting partial iodine organification defect. Thus, thyroid peroxidase (TPO) gene and DUOX2 gene were analyzed. RESULTS: There was no mutation in the TPO gene, but a novel homozygous mutation (R1110Q) in the DUOX2 gene was identified. The same heterozygous mutation was detected in her two sons and two grandchildren. This mutation was not detected in 104 control alleles and was located at a site differing from any other reported mutations in the DUOX2 gene. CONCLUSIONS: This homozygous missense mutation can be associated with thyroid dysfunction and goiter formation of an enlarged thyroid gland.

Thyroglobulin gene mutations producing defective intracellular transport of thyroglobulin are associated with increased thyroidal type 2 iodothyronine deiodinase activity.

CONTEXT: Most patients with defective synthesis and/or secretion of thyroglobulin (Tg) present relatively high serum free T3 (FT3) concentrations with disproportionately low free T4 (FT4) resulting in a high FT3/FT4 ratio. The mechanism of this change in FT3/FT4 ratio remains unknown. OBJECTIVE: We hypothesize that increased type 2 iodothyronine deiodinase (D2) activity in the thyroid gland may explain the higher FT3/FT4 ratio that is frequently observed in patients with abnormal Tg synthesis. DESIGN: We recently identified a compound heterozygous patient (patient A) with a Tg G2356R mutation and one previously described (C1245R) that is known to cause a defect in intracellular transport of Tg. In the current study, after determining the abnormality caused by G2356R, we measured D2 activity as well as its mRNA level in the thyroid gland. We also measured the thyroidal D2 activity in three patients with Tg transport defect and in normal thyroid tissue. RESULTS: Morphological and biochemical analysis of the thyroid gland from patient A, complemented by a pulse-chase experiment, revealed that G2356R produces a defect in intracellular Tg transport. D2 activity but not type 1 deiodinase in thyroid glands of patients with abnormal Tg transport was significantly higher than in normal thyroid glands, whereas D2 mRNA level in patient A was comparable with that in normal thyroid glands. Furthermore, there was a positive correlation between D2 activity and FT3/FT4 ratios. CONCLUSION: Increased thyroidal D2 activity in the thyroid gland is responsible for the higher FT3/FT4 ratios in patients with defective intracellular Tg transport.

Haplotype analysis reveals founder effects of thyroglobulin gene mutations C1058R and C1977S in Japan.

CONTEXT: Thyroglobulin (Tg) mutations were previously believed to be rare, resulting in congenital goitrous hypothyroidism. However, an increasing number of patients with Tg mutations, who are euthyroid to mildly hypothyroid, have been identified in Japan. OBJECTIVES: The purpose of this study was to investigate whether the three frequently found Tg mutations, namely C1058R, C1245R, and C1977S, were caused by a founder effect. RESULTS: We found 26 different mutations within the Tg gene in 52 patients from 41 families. Thirty-five patients were homozygous for the mutations, whereas the others were compound heterozygous. The occurrence of Tg mutation within the general Japanese population is one in 67,000. Patients with the C1245R mutation were found throughout Japan, whereas those with the C1058R mutation were confined to a small village on a southern island, and those with the C1977S mutation were restricted to a city. The eight patients with the C1058R mutation and the seven patients with the C1977S mutation all showed the same combinations of 18 single-nucleotide polymorphisms in the coding region of the Tg gene, which would appear in one in 810 million and one in 37 billion, respectively, control subjects. CONCLUSIONS: The frequently found mutations, C1058R and C1977S, were caused by founder effects. This result suggests that Tg mutations may provide a genetic basis for the cause of familial euthyroid goiter.

[In-vitro susceptibilites to levofloxacin and various antibacterial agents of 18,639 clinical isolates obtained from 77 centers in 2004].

A total of 18,639 clinical isolates in 19 species collected from 77 centers during 2004 in Japan were tested for their susceptibility to fluoroquinolones (FQs) and other selected antibiotics. The common respiratory pathogens, Streptococcus pyogenes, Streptococcus pneumoniae, Moraxella catarrhalis and Haemophilus influenzae showed a high susceptible rate against FQs. The isolation rate of beta lactamase non-producing ampicillin-resistant H. influenzae was approximately three times as large as those of western countries. Most strains of Enterobacteriaceae were also susceptible to FQs. The resistance rate of Escherichia coli against FQs has however been rapidly increasing so far as we surveyed since 1994. The FQs-resistant rate in methicillin-resistant Staphylococcus aureus (MRSA) showed approximately 90% except for 36%. of sitafloxacin while FQs-resistant rate in methicillin-susceptible S. aureus (MSSA) was around 5%. The FQs-resistant rate of methicillin-resistant coagulase negative Staphylococci (MRCNS) was also higher than that of methicillin-susceptible coagulase negative Staphylococci (MSCNS), however, it was lower than that of MRSA. In Pseudomonas aeruginosa clinical isolates, 32-34% from UTI and 15-19% of from RTI was resistant to FQs. Acinetobacter spp. showed a high susceptibility to FQs. Although FQs-resistant Neisseria gonorrhoeae have not been increased in western countries, it is remarkably high in Japan. In this survey, isolates of approximately 85% was resistant to FQs.

High incidence of thyroid cancer in long-standing goiters with thyroglobulin mutations.

In this paper, we report the high prevalence of thyroid malignancy in patients with thyroglobulin mutations in Japan. Mutations of the thyroglobulin gene cause defective thyroid hormone synthesis, resulting in congenital hypothyroidism. Since our report in 1999 on the thyroglobulin mutations C1264R and C1996S, we have identified 14 adult patients (7 males and 7 females) from 9 unrelated families. They visited hospitals for treatment of huge goiters that first appeared in childhood. Persistent growth of the thyroid gland, probably caused by thyrotropin (TSH) stimulation, partially compensated thyroid hormone production, resulting in lowered serum TSH concentrations in turn. Consequently, many patients had to undergo multiple operations. Of 11 patients who had undergone surgery, 7 had thyroid cancers. Of five patients whose thyroid tissue was available, we found a heterozygous activating mutation, either V599E or K600E, in cancerous tissue from each of 2 patients. From these observations, we conclude that goiter resulting from thyroglobulin mutations is associated with thyroid cancer.

[Medical technologist as a member of infection control team].

For the prevention of infection at institutions, an Anti-nosocomial Infection Committee or an Infection Control Team (ICT) is organized at each institution according to its scale. We report the present status of the ICT managed mainly by medical technologists engaged in microbiological examination (certified medical microbiological technologists) at Dokkyo University School of Medicine. Since this hospital is an educational hospital, the department of clinical laboratory medicine cooperates with the microbiological laboratory of the clinical laboratory in infection control education of medical workers (such as medical students, nursing students, physicians and nurses) in infection diagnosis, infection control/infection management. Since infection control is achieved by improvement in hygiene knowledge and its practice in all citizens, we also attached importance to publicity activities associated with microbiology for patients, their families, and all medical workers.

TTF-2 stimulates expression of 17 genes, including one novel thyroid-specific gene which might be involved in thyroid development.

Thyroid dysgenesis is the most frequent cause of congenital hypothyroidism, but its molecular pathophysiology is largely unknown. Our hypothesis that some genes downstream to thyroid transcription factor-2 (TTF-2) might be responsible for development of the thyroid prompted us to identify genes whose expression is stimulated by TTF-2. PCR products of cDNA clones obtained by a subtraction PCR method in TTF-2 expressing cell lines were screened with labeled cDNA by microarray analysis. We isolated 17 genes up-regulated by TTF-2, which were subsequently confirmed by quantitative reverse transcription-polymerase chain reaction (RT-PCR). One of them is a novel gene designated T1560 that showed a highly thyroid-specific expression pattern. Luciferase reporter assays showed that expression of all of the 14 genes tested was stimulated by both TTF-2 and TTF-1, another thyroid-specific transcription factor. Our results have important implications for understanding normal thyroid development as well as the molecular defects underlying thyroid dysgenesis.

Clinical significance of measurement of plasma annexin V concentration of patients in the emergency room.

Annexin V, a calcium-binding protein, is widely present in various organs and tissues. In the present study, plasma annexin V concentration was measured in 158 patients who were brought to the emergency room, including 25 patients suffering from acute myocardial infarction (AMI), 14 with cerebrovascular disease, 11 with trauma of the extremities, 11 with severe trauma associated with visceral damage, and 35 with witnessed cardiac arrest. Annexin V concentration in normal healthy individuals (n=110) was 1.9+/-0.7 ng/ml. Annexin V concentration in AMI and cardiac arrest patients was 11.0+/-4.9 and 15.3+/-7.9 ng/ml, respectively, being significantly higher than that in patients with cerebrovascular disease (5.4+/-2.7 ng/ml). The value in severe trauma patients was 15.9+/-9.4 ng/ml, being significantly higher than that in patients with trauma of the extremities (5.6+/-1.2 ng/ml). Annexin V concentrations in the cardiac arrest and AMI patients who survived more than 24 h after admission were lower than those in patients who died within 24 h after the onset of symptoms. Annexin V content in the lungs and myocardium in normal rats was extremely high in comparison to that in brain and skeletal muscle. These results suggest that the high levels of plasma annexin V in patients with AMI, cardiac arrest and severe trauma reflect the severity of damage of the myocardium and/or other visceral organs, and measurement of plasma annexin V concentration may help to assess the prognosis of patients brought to the emergency room.

[A case of pseudo-Bartter's syndrome induced by long-term ingestion of furosemide delivered orally through health tea].

A 32-year-old woman with a three-year history of muscle weakness and hypokalemia, was admitted to our hospital because of hypokalemic periodic paralysis. Clinical and laboratory findings were consistent with Bartter's syndrome. Although she denied any ingestion of diuretics substantial quantities of furosemide were detected in her urine. She had been drinking health tea which contained about 90 mg of furosemide per teabag daily for five years. Four years after discontinuation of drinking the tea, the hypokalemia was completely ameliorated, but poor renal concentration ability is still present. We conclude that is a case of pseudo-Bartter's syndrome that was caused by long-term ingestion of the health tea supplemented illegally with furosemide, and suspect that such cases may be observed more frequently than currently thought.

Does smoking cessation with varenicline worsen vascular endothelial function?

OBJECTIVES: A meta-analysis suggested that the use of varenicline, which is a partial agonist of nicotinic acetylcholine receptors and is effective in smoking cessation, increases the risk of cardiovascular events within 52 weeks of starting treatment. Defining these events as occurring during drug treatment (usually for 12 weeks) or within 30 days of discontinuation, another meta-analysis showed that the risk was statistically insignificant. In the present study, we aimed to clarify the effect of varenicline-assisted smoking cessation on vascular endothelial function assessed by flow-mediated vasodilation (FMD). DESIGN: Before-after and time-series. SETTING: Tochigi Prefecture, Japan. PARTICIPANTS: Data of 85 participants who visited nicotine-dependent outpatient services were reviewed. FMD was repeatedly measured in 33 of the 85 participants. INCLUSION CRITERIA: 20 years and older, Brinkman index ≥200, Tobacco Dependence Screener ≥5 and stated motivation to quit smoking. INTERVENTIONS: Each participant was treated with varenicline titrated up to 1.0 mg twice daily (for 12 weeks in total). PRIMARY AND SECONDARY OUTCOME MEASURES: Participants were evaluated by FMD prior to, and 3 months after, complete smoking cessation. Follow-up FMD measurements were carried out every 3 months if possible. Changes in FMD during varenicline use were also evaluated. RESULTS: FMD was significantly increased from 4.0±1.8% to 5.5±2.2% (p<0.01, n=22) 3 months after complete cessation. Although the timecourse of FMD in most of the cases showed an increase with fluctuations, there was an exceptional case where FMD decreased over the 9 months following complete cessation. Although statistically insignificant, FMD also increased during varenicline use (from 3.7±2.7% to 4.3±2.8%, n=11). CONCLUSIONS: Our observations suggest that in ceasing smokers, varenicline and smoking cessation do not lead to a worsening of the vascular endothelial function. TRIAL REGISTRATION: FK-79 (International University of Health and Welfare).