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.

[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.

[Clinical significance of apolipoprotein B-48 (apoB-48) in chronic kidney disease patients].

OBJECTIVES: Apolipoprotein B-48 (apoB-48) is a constituent of chylomicrons and their remnants, and high levels of serum apoB-48 are thought to be one of the risk factors for atherosclerosis. In the current study we examined whether serum apoB-48 level is associated with renal dysfunction. METHODS AND RESULTS: Patients were separated by eGFR into each stage of chronic kidney disease (CKD). Serum apoB-48 levels were measured by chemiluminescence enzyme immunoassay (CLEIA), and serum lipid levels were compared between each stage of CKD. Serum triglyceride (TG) levels were high at stage 4 and stage 5. Serum total cholesterol, HDL-cholesterol (HDL-C), and LDL-cholesterol (LDL-C) levels were not significantly different. Serum apoB-48 level was significantly higher at stage 4 (Median: 8.3 microg/ml) and stage 5 (9.7 microg/ml) than at stage 1(4.2 microg/ml). Serum apoB-48 levels (10.7 microg/ml) in patients undergoing hemodialysis were not significantly higher than CKD patient of nondialysis (6.9 microg/ml). CONCLUSION: Serum apoB-48 level was strongly associated with renal dysfunction. Therefore, increased serum apoB-48 concentrations may contribute to the increased risk of atherosclerosis and coronary artery disease in the CKD patients.

[The values of multi-laboratories serum values evaluation].

The members of 23 laboratories, ten clinical laboratory centers and thirteen hospital laboratories in the Kinki District participated in share their clinical laboratory data. In this joint work, we cross-checked twenty-seven serum values, and all data from the 23 laboratories well accorded; however, several values, such as urea nitrogen, calcium, and albumin needed to be standardized to share the laboratory data.

Serum apolipoprotein B-48 concentration is associated with a reduced estimated glomerular filtration rate and increased proteinuria.

AIM: Apolipoprotein B-48 (apoB-48) is a constituent of chylomicrons and their remnants (chylomicron remnants). A high concentration of serum apoB-48 is suspected to be a major risk factor for the development of atherosclerotic cardiovascular disease. Proteinuria and a reduced estimated glomerular filtration rate (eGFR) are independent risk factors for cardiovascular events and renal dysfunction. In the present study, we examined whether the serum apoB-48 concentration is associated with renal dysfunction. METHODS: A total of 264 patients was enrolled and classified into four groups according to the eGFR and level of proteinuria: a high eGFR (＞60mL/min/1.73m(2)) without proteinuria (≥1＋ by urine dipstick) (n=50); a high eGFR with proteinuria (n=75); a low eGFR (＞60mL/min/1.73m(2)) without proteinuria (n=74); and a low eGFR with proteinuria (n=65). Biochemical markers of lipid metabolism, including the fasting serum apoB-48 concentration, were compared between the four groups. RESULTS: The serum log-apoB-48 and log-apoB-48/TG levels were significantly higher in the patients with a high eGFR with proteinuria, low eGFR with proteinuria and low eGFR without proteinuria than in those with a high eGFR without proteinuria, with the most significant differences for these parameters. The eGFR was found to be significantly correlated with the log-apoB-48 and log-apoB-48/TG levels, whereas urinary protein was found to be significantly correlated with the log-apoB-48 level only. A multiple regression analysis indicated that the log-apoB-48/TG level was a significant determinant of a reduced eGFR. CONCLUSIONS: Both a low eGFR (＜60) and proteinuria (≥1＋) are independent determinants of a high apoB-48 concentration. Taken together, the present results suggest that an increased serum apoB-48 concentration contributes to an increased risk of cardiovascular events.

New homogeneous HDL-cholesterol assay without the influence of high TG sample using the selective detergent to lipoproteins.

Homogeneous HDL-cholesterol assays have been developed and used widely in routine analysis, but they have been reported to give inaccurate results in patients with hypertriglyceridemia. Recently, a new assay based on a new principle without the influence of triglycerides has also been developed and commercialized. We evaluated the basic performance of this new homogeneous HDL-cholesterol assay and compared it with the conventional polyethylene glycol/cyclodextrin-modified enzyme (PEGME) method using high-triglyceride (TG) samples (TG>8000 mg/L). For samples showing a discrepancy with the conventional method, other precipitation and ultracentrifugation (UC) methods were also used to confirm the values. This new homogeneous assay is based on the selective solubilizing effect of detergent on the different lipoproteins. First, non-HDL free cholesterol is consumed by enzyme and is cleared as a colorless reactant. Then. HDL-cholesterol is selectively solubilized by lipoprotein-specific detergent and reacted with the enzyme. As a result, the precision of this new homogeneous assay was good (CV<2%) over the wide range, and the measurement range was 0 to 2000 mg/L. This method correlated well with the PEGME method, which is a conventional method for normolipidemic samples (y=0.97x-3.1, r=0.994, n=424). It also correlated well with the UC method (y=0.99x+0.3, r=0.989, n=53). Fourteen high-TG samples showed different results from those obtained by the PEGME method. Among these samples, one contained abnormal lipoproteins (probably due to the influence of drug therapy) and gave a significantly different result from that obtained by the PEGME method. However, the values obtained by other methods (precipitation and ultracentrifugation) agreed well with those obtained by this new method. In conclusion, this method shows a good basic performance and is useful for high-TG samples without any interference. Therefore, it is considered to be very practical for a routine test.