Urinary mulberry bodies as a potential biomarker for early diagnosis and efficacy assessment of enzyme replacement therapy in Fabry nephropathy.

BACKGROUND: The inability of enzyme replacement therapy (ERT) to prevent progression of Fabry nephropathy (FN) in the presence of >1 g/day proteinuria underscores the necessity of identifying effective biomarkers for early diagnosis of FN preceding proteinuria. Here we attempted to identify biomarkers for early detection of FN. METHODS: Fifty-one Fabry disease (FD) patients were enrolled. Urinary mulberry bodies (uMBs) were immunostained for globotriaosylceramide (Gb3) and renal cell markers to determine their origin. The association between semiquantitative uMB excretion and the histological severity of podocyte vacuolation was investigated in seven patients using the vacuolated podocyte:glomerular average area ratio. The association between the semiquantitative estimate of uMB excretion and duration of ERT was analyzed. A longitudinal study was conducted to assess the effect of ERT on uMB excretion. RESULTS: Thirty-two patients (63%) had uMBs, while only 31% showed proteinuria. The uMBs were positive for Gb3, lysosomal-associated membrane protein 1 and podocalyxin, suggesting they were derived from lysosomes with Gb3 accumulation in podocytes. We observed more severe podocyte vacuolation with increased uMB excretion (P = 0.03 for trend); however, the same was not observed with increased proteinuria. The percentage of patients with substantial uMB excretion increased with shorter ERT duration (P = 0.018). Eighteen-month-long ERT reduced uMB excretion (P = 0.03) without affecting proteinuria. CONCLUSIONS: uMB excretion, implying ongoing podocyte injury, preceded proteinuria in most patients. Semiquantitative uMB estimates can serve as novel biomarkers for early FN diagnosis and for monitoring the efficacy of FD-specific therapies.

Gene polymorphisms of VEGF and VEGFR2 are associated with the severity of Hashimoto's disease and the intractability of Graves' disease, respectively.

Vascular endothelial growth factor (VEGF) is one of main regulators of angiogenesis that functions by binding to its receptors, including VEGF receptor (VEGFR) 2. There are few data available regarding the association between VEGF and VEGFR polymorphisms and the susceptibility to and prognosis of autoimmune thyroid diseases (AITDs). To elucidate this association, we genotyped four functional VEGF and two VEGFR2 polymorphisms and measured serum VEGF levels. In the four functional VEGF polymorphisms, the frequencies of the I carrier and I allele of VEGF -2549 I/D, which has lower activity, were higher in patients with severe HD than in those with mild HD. In the two functional VEGFR2 polymorphisms, the frequency of the rs2071559 CC genotype, which has higher activity, was higher in patients with intractable GD than in controls, and the proportion of GD patients with larger goiters was higher in those with the CC genotype. Moreover, the frequency of the rs1870377 TT genotype with higher activity was higher in patients with intractable GD than in those with GD in remission. Combinations of VEGF and VEGFR2 polymorphisms with stronger interactions were associated with the intractability of GD. Serum VEGF levels were higher in HD and AITD patients than those in controls. In conclusion, VEGF polymorphisms with lower activity were associated with the severity of HD, while VEGFR2 polymorphisms and the combinations of VEGF and VEGFR2 polymorphisms, which have stronger interactions, were associated with the intractability of GD. VEGF and VEGFR2 polymorphisms were associated with HD severity and GD intractability, respectively.

Association of IL6 gene methylation in peripheral blood cells with the development and prognosis of autoimmune thyroid diseases.

Autoimmune thyroid diseases (AITDs), including Hashimoto's disease (HD) and Graves' disease (GD), are archetypes of organ-specific autoimmune diseases, but the prognosis of patients with AITD varies. Autoimmune diseases, including AITDs, are believed to develop in response to both genetic and environmental factors. Interleukin (IL)-6 plays a major role in B cell differentiation and T cell proliferation, and methylation of the IL6 gene is associated with IL-6 production. To clarify the role of IL6 gene methylation in the pathogenesis and prognosis of AITDs, we measured the methylation levels of -666, -664, -610, -491 and -426 CpG sites in the IL6 gene. We measured the methylation levels of 5 CpG sites in 29 patients with HD, 31 patients with GD and 16 healthy volunteers using pyrosequencing. The methylation level at each of the -664, -491 and -426 CpG sites was negatively correlated with the age at the time of sampling. Multiple regression analysis indicated that patients with HD, including severe or mild HD, showed higher methylation levels at the -426 CpG site than control subjects. Patients with intractable GD showed lower methylation levels at the -664 and -666 CpG sites than patients with GD in remission. In conclusion, IL6 gene methylation levels were related to the susceptibility to HD and the intractability of GD.

Methylation levels of the TNFA gene are different between Graves' and Hashimoto's diseases and influenced by the TNFA polymorphism.

Graves' disease (GD) and Hashimoto's disease (HD) are different pathological types of autoimmune thyroid diseases (AITDs). However, the epigenetic differences between these diseases have not been elucidated. DNA methylation is one of the primary epigenetic modifications that reflect environmental influences on gene expression. In this study, we evaluated the methylation status of six CpG sites in the TNFA promoter using pyrosequencing and analyzed the data in combination with functional polymorphisms (-1031 T/C and +123 C/T) in the TNFA gene to clarify the role of gene methylation on the prognosis of AITDs. We examined the methylation pattern in 52 patients with GD, 60 patients with HD, and 29 healthy controls by pyrosequencing. Additionally, we also genotyped the polymorphisms from 163 patients with GD, 152 patients with HD, and 94 healthy controls using the restriction fragment length polymorphism (RFLP) method. Each proportion of subjects with low methylation of the -72 CpG site (≤11.9%), low methylation of the -49 CpG site (≤15.5%), and low methylation of the -38 CpG site (≤8.9%) was significantly increased in the groups with high concentration of TNF-α (≥0.134 pg/mL). The methylation level of the -72 CpG site was significantly higher in GD cases (10.7 ± 4.9%) than in healthy controls (6.8 ± 3.9%). The methylation level of the -49 and -38 CpG sites were significantly higher in patients with GD in remission (20.5 ± 9.5%, 17.6 ± 8.0%, respectively) than in healthy controls (13.0 ± 7.6%, 7.9 ± 7.3%, respectively). The frequency of the TNFA - 1031C carrier (CT + CC) is correlated with higher TNF-α production and was significantly higher in GD (35.0%) and HD (39.5%) cases than in controls (19.1%). In the subjects with the TNFA - 1031C carrier (CT + CC), the methylation level of the -72 CpG site was significantly higher in GD (11.5 ± 5.7%) than in HD (6.0 ± 3.4%). However, there was no difference between GD and HD in patients with the TT genotype. Cumulatively, our data indicate the methylation levels of CpG sites in the TNFA gene may be related to the difference between GD and HD in AITDs and may be influenced by the TNFA gene polymorphism.

Comparison of the Temperature Influence on the Activity of Currently Available Procalcitonin Reagents.

BACKGROUND: Procalcitonin (PCT) is a stable biomarker for bacterial infections; however, limited data is available on new trivalent reagents. We evaluated temperature influence on the activity of PCT reagents. METHODS: Using both conventional and trivalent reagents, we measured PCT levels of 30 clinical samples, stored residuum at refrigerator (4°C) and room temperature (24°C), and reexamined it after 24 hours. We defined a reduction rate as a percentage of PCT level at 24 hours compared to that after defrost and evaluated a ratio of reduction rate in 4°C to that in 24°C. RESULTS: The reduction rate at room temperature decreased significantly compared to that in the refrigerated condition for all the reagents examined (p < 0.001). In addition, the ratio of reduction rate between the conventional and trivalent reagents showed a significant difference (p < 0.001). CONCLUSIONS: The serum PCT levels significantly decrease at room temperature, particularly when using newer trivalent reagents.

Evaluation of the highly sensitive chemiluminescent enzyme immunoassay "Lumipulse HBsAg-HQ" for hepatitis B virus screening.

BACKGROUND: Ongoing efforts in the development of HBsAg detection kits are focused on improving sensitivity and specificity. The purpose of this study was to evaluate an improved, highly sensitive quantitative assay, "Lumipulse HBsAg-HQ", a chemiluminescent enzyme immunoassay designed for a fully automated instrument, the "Lumipulse G1200". METHODS: Serum samples for reproducibility, dilution, correlation, sensitivity, and specificity studies were obtained from patients at the Osaka University Hospital. Seroconversion and sensitivity panels were purchased from a commercial vender. Subtype, sensitivity panels, and HBsAg recombinant proteins with one or two amino acid substitutions were prepared in-house. RESULTS: The coefficients of variation for the low, medium, and high concentration samples ranged from 1.93 to 2.55%. The HBsAg-HQ reagent for dilution testing showed good linearity in the 0.005-150 HBsAg IU/mL range and no prozone phenomenon. All 102 HBV carrier samples were positive by HBsAg-HQ, while other commercial reagents showed one or more to be negative. In the seroconversion panel, the 14-day blood sample was positive. The sensitivity against HBsAg-HQ "ad" and "ay" subtypes was 0.025 ng/mL. Comparisons among the HBsAg-HQ, HISCL, and Architect HBsAg reagents were performed using the Bland-Altman plot. Specificity for 1000 seronegative individuals was 99.7%. HBsAg-HQ detected 29 positive serum among 12 231 routinely obtained serum samples, which showed concentrations of 0.005-0.05 HBsAg IU/mL. CONCLUSIONS: According to these results, the Lumipulse HBsAg-HQ assay, with a highly sensitive limit of detection of 0.005 IU/mL, may facilitate the development of a better management strategy for a considerable proportion of infected patients.

Involvement of genes encoding apoptosis regulatory factors (FAS, FASL, TRAIL, BCL2, TNFR1 and TNFR2) in the pathogenesis of autoimmune thyroid diseases.

Apoptosis is necessary for the maintenance of self-tolerance by eliminating autoreactive immune cells in the periphery. To clarify the association between the pathogenesis of autoimmune thyroid disease (AITD) and genes encoding apoptosis regulatory factors, we genotyped the FAS -1377G/A, -670A/G, FASL -844C/T, TRAIL -716C/T, BCL2 -938C/A, +127G/A, TNFR1 -383A/C and TNFR2 +676T/G polymorphisms. The frequencies of the FASL -844CC and BCL2 -938AA genotypes were significantly lower in AITD patients than in control subjects (P=0.0101 and 0.0307, respectively). The frequency of the TNFR2 +676TT genotype was significantly lower in Graves' disease (GD) patients than in controls (P=0.0284). The serum sFasL level was significantly higher in GD and Hashimoto's disease (HD) patients than in control subjects (P=0.0003 and 0.0017, respectively). The serum sFasL levels in control subjects were significantly lower than those in intractable GD, GD in remission, and HD without treatment (P=0.0310, 0.0007 and 0.0002, respectively). The serum sFasL levels in HD with treatment were significantly lower than those in HD without treatment (P=0.0490). The polymorphisms in genes encoding apoptosis regulatory factors (FASL, BCL2) and serum levels of sFasL may be associated with immune dysregulation.

Association of polymorphisms in the ICOS and ICOSL genes with the pathogenesis of autoimmune thyroid diseases.

The prognosis of autoimmune thyroid diseases (AITDs), including Graves' disease (GD) and Hashimoto's disease (HD), varies among patients. Inducible co-stimulator (ICOS) (CD278) and co-stimulator ligand (ICOSL) (CD275) are important costimulatory molecules. Their interactions play important roles in immune regulation and the pathogenesis of autoimmune diseases through tuning T cell activation, differentiation and function. To clarify the association between ICOS-ICOSL signals and AITD, we genotyped single-nucleotide polymorphism (SNP)1 and SNP2 in the ICOS gene and SNP1, SNP2 and SNP3 in the ICOSL gene in 239 HD patients, 232 GD patients, and 129 healthy volunteers (control subjects). There were no differences in genotype and allele frequencies among the three groups, although the frequencies of the AA genotype and A allele of ICOSL SNP2 (rs15927) were slightly, but not significantly, higher in patients with GD, intractable GD, and severe HD than in controls. The mRNA levels of ICOSL were also slightly, but not significantly, lower in individuals with the AA genotype of ICOSL SNP2 than in those with the AG+GG genotypes. In conclusion, the ICOS and ICOSL SNPs examined in this study do not have an apparent effect on the disease susceptibility and prognosis of AITDs.

Latex-enhanced turbidimetric immunoassay for everolimus in whole blood using the Nanopia TDM everolimus assay with the JCA-BM6010 automatic analyzer.

BACKGROUND: Everolimus is a novel proliferation signal inhibitor used in immunosuppressive therapies for the prevention of acute and chronic rejection. It is an immunosuppressant requiring routine monitoring in whole blood. We evaluated analytical performance of the Nanopia TDM Everolimus assay kit for the measurement of everolimus in whole blood. METHODS: Whole blood samples from patients receiving immunosuppressive therapy with everolimus after heart transplantation were used, and everolimus concentrations were measured by liquid chromatography combined with mass spectrometric detection and fluorescence polarization immunoassay and Nanopia TDM Everolimus assay. RESULTS: The within-assay coefficient of variation was 4.0%-6.8% (n = 20) at everolimus concentrations of 4.4-15.7 ng/mL, whereas the day-to-day coefficient of variation ranged from 3.6% to 5.4% at everolimus concentrations of 4.8-15.9 ng/mL. The limit of quantitation was 1.5 ng/mL. Calibration curves were stable for at least 14 days. The presence of conjugated bilirubin, unconjugated bilirubin, lipemic material, rheumatoid factor, and variation of the hematocrit did not affect this assay system. There was a strong positive correlation between values obtained with the Nanopia TDM Everolimus assay kit and the results of liquid chromatography combined with mass spectrometric detection (y = 0.960x + 0.312 ng/mL; r = 0.946; n = 91), as well as with data from the fluorescence polarization immunoassay (y = 0.966x - 0.440 ng/mL; r = 0.942; n = 91). CONCLUSIONS: The Nanopia TDM Everolimus assay showed good analytical performance. These results indicate that the Nanopia TDM Everolimus assay may be suitable for routine clinical use.

[Evaluation of the Creatine Kinase MB Activity Assay Kit].

CK-MB activity, which is measured by the immunoinhibition method, is an important marker for use in the diagnosis of acute coronary syndromes. In the present study, we evaluated the basal performance of a recently improved CK-MB activity kit, "L-system CK-MB," in which the activity of mitochondrial CK subunits is inhibited. Within-run and between-day precision were in the range of 1.9-2.3% and 3.2-6.0%, respectively. Diluted linearity and limit of detection were determined to be 600 U/L and 0.8 U/L, respectively. The use of L-system CK-MB allowed the inhibition of the activity of 98.1% of sarcomeric mitochondrial CK, 97.7% of ubiquitous mitochondrial CK, and 99.9% of CK-MM. The correlation coefficient (r) between CK-MB activity and CK-MB protein was 0.968. However, we found 4 cases showing CK-MB activity significantly higher than the protein concentration. Increased CK-BB activity was detected by electrophoresis in these cases. In some patients with malignant tumors, the presence of CK-immunoglobulin complex also lead to elevated CK-MB concentrations. Thus, the discrepancy in the CK-MB activity and the protein concentration may be caused by the presence of CK-BB and/or CK-immunoglobulin complex. More attention needs to be focused on samples with high CK-MB protein concentrations, especially when the CK-MB/CK ratio is high.

Preparation of thyroid follicular cells for mRNA quantification after fluorescence-activated cell sorting.

We established a novel method to analyze cells collected by fluorescence-activated cell sorting (FACS) named mRNA quantification after FACS (FACS-mQ) in which cells are labeled with a fluorescence dye in a manner that minimizes RNA degradation, and then cells sorted by FACS are examined by analyzing their gene expression profile. In order to analyze cells using FACS-mQ, it is essential to prepare single-cell suspensions without RNA degradation. We found that a new tissue preservation medium, ThelioKeep™, which contains epigallocatechin-3-gallate (EGCG), was suitable for preservation of thyroid tissues. The aim of this study was to establish a cell dispersion method of thyroid follicular cells using ThelioKeep™. We compared the efficiency of cell dispersion between the two methods, the conventional cold pre-incubation method and the ThelioKeep™ method; then we determined if cells obtained by the ThelioKeep™ method were suitable for FACS-mQ analysis. We found that a larger number of cells were recovered using ThelioKeep™ than using the conventional cold pre-incubation method. Furthermore, cell viability was higher with the ThelioKeep™ method than with the cold pre-incubation method. Thyroid cells collected by this method were analyzed by FACS-mQ. A clear shift in flow cytometry analysis was observed when cells were stained with an anti-thyroglobulin or anti-thyroid transcription factor-1 antibody. After sorting, the same copy number of ACTB mRNA was detected in thyroid cells as in an anaplastic carcinoma cell line, 8305C. These findings imply that preparation of thyroid cells using the present method is suitable for FACS-mQ analysis.

[Evaluation of the latex turbidimetric immunoassay for CK-MB protein].

CK-MB protein is an important marker for the diagnosis of acute coronary syndromes. In the present study, we evaluated the basal performance of recently developed CK-MB mass kit "L-type wako CK-MB mass". Within-run and between-day precision were obtained with 1.4-4.7% and 2.7-5.2%, respectively. Diluted linearity and lower limit of detection were obtained with 180.0 ng/mL and 2.1 ng/mL, respectively. Zone phenomenon was able to detect until 25,600.0 ng/mL. Analysis of interferent showed that only CK-BB positively influenced the assay results. CK-MB protein levels decreased to 82% at 72 hours in the room temperature, but it was stable at 4 degrees C and -20 degrees C. The correlation coefficient(r) between this assay and conventional assay was 0.999. However, discrepancy of the two cases was observed in the comparison between the two methods. In the case 1, CK isoenzyme analysis using electrophoresis indicated that CK-MB was not present and absorption test showed a 68% absorption effect of CK-MB protein values not by anti human IgG, anti human IgA, and animal serums, but anti human IgM. In the case 2, CK isoenzyme analysis indicated that there is not only CK-MB but CK-BB. CK-MB protein values between the two methods were fitted after decreasing CK-BB. Thus, Value discrepancy for CK-MB protein was resulting from IgM and CK-BB. We have to pay attention to such phenomenon when detecting an unlikely higher levels that could not be explained by clinical information.

Genes that characterize T3-predominant Graves' thyroid tissues.

OBJECTIVE: 3,5,3'-Triiodothyronine (T(3))-predominant Graves' disease is characterized by the increasing volume of thyroid goiter resulting in poor prognosis. Although type 1 and type 2 iodothyronine deiodinases (DIO1 and DIO2 respectively) are known to be overexpressed in the thyroid tissues of T(3)-predominant Graves' disease, the pathogenesis of this disease is still unclear. The aim of our study is to identify genes that characterize T(3)-predominant Graves' disease tissue in order to clarify the molecular mechanism of this disease. DESIGN AND METHODS: mRNAs from two thyroid tissues of both typical T(3)-predominant and common-type Graves' disease were analyzed with DNA microarrays with probes for 28 869 genes. Genes identified to be differentially expressed between the two groups were further analyzed in the second and third screenings using 70 Graves' thyroid tissues by real-time quantitative RT-PCR. RESULTS: Twenty-three candidate genes were selected as being differentially expressed in the first screening with microarrays. Among these, seven genes, leucine-rich repeat neuronal 1 (LRRN1), bone morphogenetic protein 8a (BMP8A), N-cadherin (CDH2), phosphodiesterase 1A (PDE1A), creatine kinase mitochondrial 2 (CKMT2), integrin beta-3 (ITGB3), and protein tyrosine phosphatase non-receptor type 4 (PTPN4), were confirmed to be differentially expressed in DIO1 or DIO2 over- and underexpressing Graves' tissues. CONCLUSIONS: These genes are related to the characteristics of T(3)-predominant Graves' disease, such as high titer level of serum anti-TSH receptor antibody, high free T(3) to free thyroxine ratio, and a large goiter size. They might play a role in the pathogenesis of T(3)-predominant Graves' disease.

[FACS-mQ as a powerful clinical test in the cancer stem cell era].

Stem cells are pluripotent and self renewing, and possess an ability to differentiate into the various cell types of a particular tissue. In cancer tissues, existence of cells showing biological similarities with stem cells, named cancer stem cells (CSC), are known to regulate the growth of the tissue and determine its prognosis. Stem cells and CSCs usually exist as minor populations of cells in a tissue. Detection and analysis of these cells are usually laborious even using fluorescence activated cell sorting (FACS) with the conventional protocols. Considering these drawbacks, we developed a novel analytical method named mRNA quantification after FACS (FACS-mQ). In FACS-mQ, cells are labeled with a fluorescent dye in a manner that minimizes RNA degradation, then cells sorted by FACS are examined by analyzing their gene expression profile. We established protocols to obtain single cells from clinical samples for flow cytometry analysis. Further, we performed FACS-mQ analysis using fluorescence-labeled antibodies, cRNA probes and locked nucleic acid (LNA) probes. Evident decrease of intracellular RNAs did not observed in FACS-mQ using immunocytochemistry. Approximately 60% of intracellular RNA was preserved after in situ hybridization using cRNA probes. These RNAs from a small number of sorted cells were suitable for quantitative analysis to establish gene expression profiles. FACS-mQ does not require laborious and time-consuming procedures; thus, it is expected to facilitate research on stem cells or cancer stem cells.

Associations between autoimmune thyroid disease prognosis and functional polymorphisms of susceptibility genes, CTLA4, PTPN22, CD40, FCRL3, and ZFAT, previously revealed in genome-wide association studies.

PURPOSE: Genome-wide association studies have revealed several susceptibility genes among patients with autoimmune thyroid disease (AITD), including CTLA4, PTPN22, FCRL3, and ZFAT. However, any possible association between these genes and AITD prognosis remains unknown. The objective of this study was to identify associations between polymorphisms of these genes and AITD prognosis. METHODS: We genotyped functional polymorphisms, including CTLA4 CT60, CTLA4 +49A/G, CTLA4 -1147C/T, CTLA4 -318C/T, PTPN22 -1123C/G, PTPN22 SNP37, CD40 -1C/T, FCRL3 -169C/T, ZFAT Ex9b-SNP10, and ZFAT Ex9b-SNP2, in 197 AITD patients carefully selected from 456 registered AITD patients, and 86 control subjects. The restriction fragment length polymorphism method was used for genotyping. RESULTS: The CD40 -1CC genotype and C allele were significantly more frequent in patients with Graves' disease (GD) in remission than in those with intractable GD (P = 0.041 and P = 0.031, respectively). The FCRL3 -169TT genotype was significantly less frequent in patients with intractable GD than in those with GD in remission (P = 0.0324). For a ZFAT Ex9b-SNP10 polymorphism, the TT genotype and T allele were significantly more frequent in patients with severe Hashimoto's disease (HD) than in those with mild HD (P = 0.0029 and P = 0.0049, respectively). For a CTLA4 CT60 polymorphism, the antithyrotropin receptor antibody levels at the onset of GD were significantly higher in those with the GG genotype than in those with other genotypes (P = 0.0117). CONCLUSIONS: CD40 and FCRL3 gene polymorphisms were associated with GD intractability, and ZFAT polymorphism was associated with HD severity but not its development.

Measurement of TFF3 mRNA in aspirates from thyroid nodules using mesh filtration: the first clinical trial in 130 cases.

Measurement of gene expression levels in thyroid tumor cells in aspirates was difficult because it is interfered with peripheral blood cells or infiltrating lymphocytes. In this study, we established a novel method to separate thyroid tumor cells from blood cells efficiently with mesh filtration. The expression level of trefoil factor 3 (TFF3) mRNA was estimated using LGALS3 mRNA as an internal control (T/G ratio) in 148 preoperative thyroid aspirates. Intra-assay coefficients of variation (CV) of T/G ratio for high, moderate, and low samples were 6.5%, 2.5%, and 9.7%, respectively, and inter-assay CV for high, moderate, and low samples were 27.7%, 21.9%, and 38.2%, respectively. Nondiagnostic samples in terms of T/G ratio and cytology were 12.2% and 16.9%, respectively. We observed no interference with the data by contaminating blood cells. Among these patients, 12 patients received more than two repeated aspirations. We did not observe a marked day-to-day variation except in two cases. All 13 preoperative aspirates diagnosed as malignant by cytology showed an extremely low T/G ratio, whereas 93 aspirates diagnosed as benign by cytology showed extremely varied T/G ratios and 21.5% of them showed a T/G ratio below the cut-off value. Eleven cases underwent surgery. All nodules showing a low T/G ratio were diagnosed as papillary carcinoma by pathological diagnosis. However, one nodule diagnosed as follicular adenoma after surgery showed a high T/G ratio. Our present method may be a promising preoperative test for measuring mRNAs in thyroid aspirates.

[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.

Establishment of chemiluminescence enzyme immunoassay for apolipoprotein B-48 and its clinical applications for evaluation of impaired chylomicron remnant metabolism.

BACKGROUND: Apolipoprotein B-48 (apoB-48) is a constituent of chylomicron remnants synthesized in the small intestines. The serum concentration of apoB-48 at fasting has been reported to be a marker of postprandial hyperlipidemia, a presumed risk factor for atherosclerosis. METHODS: We evaluated the basal performance of a recently developed chemiluminescent enzyme immunoassay (CLEIA). We also examined the correlations between serum apoB-48 concentrations and other lipid concentrations or life style patterns, including smoking and drinking. We analyzed the data of 273 clinical samples by multiple regression analysis to examine the influence of other serum lipid values, age, sex, smoking, drinking status and BMI on serum apoB-48 values. RESULTS: Within-run and between-run precision was obtained with 1.7-2.7% and 1.2-7.3%, respectively. The correlativity of enzyme-linked immunosorbent assay was correlation coefficient r=0.953, and regression y=1.02×-1.59. Serum apoB-48 concentrations were higher in males than in females, and were correlated with the status of smoking as well as with remnant-like particle-cholesterol (RLP-C) concentrations. Patients with the metabolic syndrome showed higher values of serum apoB-48 compared with control subjects. CONCLUSION: Serum apoB-48 measurement by CLEIA was satisfactory for clinical use to assess abnormalities in the chylomicron remnant metabolism.