Evaluation of a candidate reference measurement procedure for serum free testosterone based on ultrafiltration and isotope dilution-gas chromatography-mass spectrometry

BACKGROUND: To assess the analytical validity of free testosterone (FTe) measurements, a reference measurement procedure (RMP) is required. For steroids, isotope dilution-mass spectrometry is accepted as state-of-the-art technology. Because FTe is defined as the hormone fraction in serum water in equilibrium with the protein-bound fraction, the RMP should include a physical separation step. The use of equilibrium dialysis (ED) or ultrafiltration (UF) is advocated. Our objective was to develop such a candidate RMP. METHODS: We selected UF combined with isotope dilution-gas chromatography-mass spectrometry (ID-GC/MS) for direct measurement of Te in the ultrafiltrate. After optimization of the UF process, the complete procedure was validated by use of split-sample comparisons with indirect ED (iED) and symmetric dialysis (SyD). RESULTS: The candidate RMP gave maximum within-day, between-day, and total CVs of 3.0%, 3.1%, and 4.3%. The Deming regression equations for the respective method comparisons were: UF-ID-GC/MS = 0.98(iED) - 53 pmol/L (r = 0.94; S(y|x)= 42 pmol/L) and UF-ID-GC/MS = 0.92(SyD) + 21 pmol/L (r = 0.97; S(y|x)= 31 pmol/L). CONCLUSIONS: We achieved the objective of a state-of-the-art candidate RMP, which agreed well with iED and SyD. However, we also demonstrated that a degree of discordance remains, which may require a decision from an authoritative organization on the recommended procedure to measure free hormone concentrations.     

Neutralizing antibodies to interferon beta in multiple sclerosis: analytical evaluation for validation of a cytopathic effect assay

BACKGROUND: Recent guidelines have recommended the use of validated assays for the measurement of neutralizing antibodies (NABs) to interferon beta (IFNbeta) in patients with multiple sclerosis (MS). In an attempt of validation, we studied the analytical performance of a bioassay based on antiviral cytopathic effect (CPE) using WISH cells and the vesicular stomatitis virus (WISH/VSV CPE). METHODS: NAB titres measured with the WISH/VSV CPE assay in 63 sera from IFNbeta-treated MS patients were compared to those obtained with the reference CPE method using A549 cells and the encephalomyocarditis virus. Binding antibodies (BABs) were measured using a capture ELISA as a screening test for NABs. RESULTS: No false-negative BAB was obtained in our patients. The between-run coefficients of variation (CVs) determined with log10 titres of the NIH anti-IFNbeta (G038-501-572) yielded good results (相似文献   

Importance of validation of immunoassays for intact proinsulin.

The aim of this study was to compare results obtained from two commercially available immunoassay kits for intact proinsulin. Fasting and post-prandial samples were obtained from both healthy subjects and patients with type 2 diabetes mellitus and assays were carried out according to the manufacturers' instructions. Coefficient of variation of the duplicates in both assays was acceptable with the MLT Intact Proinsulin assay giving slightly better overall precision. Regression analysis indicated a good correlation between the assays (r=0.97), however, a procedure better designed to compare analytical methods demonstrated a considerable lack of agreement for some samples. Dilution of samples in the Dako assay greatly affected the results when compared to samples assayed undiluted, whereas in the MLT assay, dilution of samples produced the expected results. Repeat comparison, assaying samples neat in the MLT assay and diluted 1:5 in the Dako assay, resulted in a considerable improvement in the agreement between the Dako and MLT assays. This study underlines the importance of the use of validation procedures which demonstrate quantitative analytical recoveries from a variety of specimens over the working range of the assay method in question.     

三种稀释蝰蛇毒磷脂时间试验检测狼疮抗凝物分析性能评估

摘要:目的?对3种稀释蝰蛇毒磷脂时间试验(dRVVT)的狼疮抗凝物(LA)检测方法进行性能评价，比较不同系统间检测结果的一致性，并初步探讨不同检验流程对检测结果的影响。方法?按照美国临床和实验室标准协会(CLSI)文件及临床血液学检测常规项目分析质量文件(WS/T 406—2012)，对3种LA检测方法的精密度、携带污染率进行评价，验证3种方法检测LA的参考区间(RI)/cut-off值，并分析检验结果一致性和不同检验流程对检测结果的影响。结果?3种dRVVT检测方法重复性以CV表示为0.44%～1.69%；期间精密度以CV表示，为1.43%～2.43%。3种检测方法携带污染率绝对值<3.00%。系统1和2的RI验证通过，系统3验证未通过。3种不同系统筛选试验、确认试验及比值间结果差异有统计学意义(P=0.000)。3种不同系统阴阳性判断结果间差异有统计学意义(χ2=11.333，P=0.000)。系统3以说明书推荐流程分析结果，与筛选/确认比值(R)或标准化比值(NR)结果表示方式相比，53例阴性结果中2例为假阴性。结论?3种dRVVT检测系统性能良好，适于临床常规使用。但各实验室应验证或建立适合本实验室的RI/cut-off值，确定合理的检测流程，关注检测分析前中后影响因素，合理解释检测结果。     

Analytical performance and clinical efficacy of three routine procedures for LDL cholesterol measurement compared with the ultracentrifugation-dextran sulfate-Mg(2+) method

The diagnosis and management of adults with hypercholesterolemia in the US are largely based on low-density lipoprotein cholesterol (LDL-C) concentration. In order to classify someone correctly into the National Cholesterol Education Program cut-points, LDL-C must be measured with a total error of 相似文献   

Development and evaluation of assays for the determination of total and pancreatic amylase at 37°C according to the principle recommended by the IFCC

Objectives: The aim of our study was a) to optimize assays for measurement of total (T-) and pancreatic (P-)amylase at 37°C based on the principle recommended by the IFCC at 30°C, b) to evaluate the analytical performance of these assays in a multicentric study and c) to establish reference intervals for serum and urine for either method.Methods: Optimized conditions for 37°C were elaborated with regard to substrate concentration, pH, inorganic additives and glucosidase activity. The cleavage pattern of the EPS substrate was studied by HPLC. Liquid ready-to-use reagents for T- and P-amylase were provided to six European laboratories.Results: The assays showed good performance characteristics (median intraassay CVs 1.0% for T- and 1.3% for P-amylase, median interassay CVs 3.0% for either assay, dynamic range 15-fold URL for T- and 30-fold for P-amylase), high correlation with the previous EPS methods (r > 0.996, slope 0.43, intercept < 5 U/L) in serum, heparin plasma and urine and good analytical specificity of the P-amylase assay (residual S-amylase activity 2.4%). Serum reference ranges were found to be 28 to 100 U/L for T- and 13 to 53 U/L for P-amylase (n = 775); URLs in urine were estimated as 490 U/L or 280 U/g creatinine for males and 450 U/L or 380 U/g creatinine for females with total amylase.Conclusion: We believe that these assays based on the 30°C IFCC recommendation represent a further improvement in amylase methodology at 37°C and merit broad application in clinical routine.     

Quantitative, wide-range, 5-minute point-of-care immunoassay for total human chorionic gonadotropin in whole blood

BACKGROUND: Human chorionic gonadotropin (hCG) is among the most common analytes available for point-of-care immunotesting, with most assays currently based on simple manual assay devices. However, as the importance of good analytical performance of rapid assays is increasingly emphasized, more sophisticated immunoassay techniques are needed to meet the future challenges of rapid yet quantitative POC testing. METHODS: We developed a simple, dry-reagent, all-in-one immunoassay for the quantitative measurement of hCG in whole blood, plasma, or serum. The noncompetitive assay equally measures intact, nicked, and hyperglycosylated hCG as well as nonnicked and nicked hCG beta-subunit with a rapid and simple procedure consisting of a 5-min, one-step incubation and, subsequent to washing, the measurement of time-resolved fluorescence directly from a wet well surface. RESULTS: The assay had a detection limit (background + 3 SD) of 0.4 IU/L hCG. The within-run CV was <15% down to 2 IU/L, and the assay was linear to 6000 IU/L. The within- and between-run CVs in heparinized whole blood and plasma were 相似文献   

Development and evaluation of assays for the determination of total and pancreatic amylase at 37 degrees C according to the principle recommended by the IFCC

OBJECTIVES: The aim of our study was a) to optimize assays for measurement of total (T-) and pancreatic (P-)amylase at 37 degrees C based on the principle recommended by the IFCC at 30 degrees C, b) to evaluate the analytical performance of these assays in a multicentric study and c) to establish reference intervals for serum and urine for either method. METHODS: Optimized conditions for 37 degrees C were elaborated with regard to substrate concentration, pH, inorganic additives and glucosidase activity. The cleavage pattern of the EPS substrate was studied by HPLC. Liquid ready-to-use reagents for T- and P-amylase were provided to six European laboratories. RESULTS: The assays showed good performance characteristics (median intraassay CVs 1.0% for T- and 1.3% for P-amylase, median interassay CVs 3.0% for either assay, dynamic range 15-fold URL for T- and 30-fold for P-amylase), high correlation with the previous EPS methods (r > 0.996, slope 0.43, intercept < 5 U/L) in serum, heparin plasma and urine and good analytical specificity of the P-amylase assay (residual S-amylase activity 2.4%). Serum reference ranges were found to be 28 to 100 U/L for T- and 13 to 53 U/L for P-amylase (n = 775); URLs in urine were estimated as 490 U/L or 280 U/g creatinine for males and 450 U/L or 380 U/g creatinine for females with total amylase. CONCLUSION: We believe that these assays based on the 30 degrees C IFCC recommendation represent a further improvement in amylase methodology at 37 degrees C and merit broad application in clinical routine.     

Partly nonparametric approach for determining the limit of detection

BACKGROUND: According to recent International Organization for Standardization (ISO) standards, the limit of detection (LoD) of an assay should be estimated taking both type I (alpha) and II (beta) errors into account. The suggested procedure, however, supposes gaussian distributions of both blank and sample measurements and a linear calibration curve. In clinical chemistry, asymmetric, nongaussian blank distributions are common, and the calibration curve may be nonlinear. We present a partly nonparametric procedure that takes these aspects into account. METHODS: Using theoretical distribution models and simulation studies, we developed a LoD estimation procedure suitable for the field of clinical chemistry that is partly based on nonparametric statistics. RESULTS: For sample size n, the nonparametrically determined 95th percentile of the blank measurements obtained as the value of the [n(95/100) + 0.5]th ordered observation defines the limit for results significantly exceeding zero [limit of blank (LoB)]. The LoD is the lowest value that is likely to yield a result exceeding the LoB. LoD is estimated as: LoB + cbeta x SDS, where SDS is the analytical SD of a sample with a low concentration; cbeta = z(1 - beta)/[1 - 1/(4 x f)]; z(1 - beta) is the standard normal deviate; and f is the number of degrees of freedom for estimation of SD(S). c(beta) is approximately equal to 1.65 for a type II error of 5%. Approaches and needed tabular values for calculation of confidence limits are presented as well as sample size. Worked examples are given to illustrate estimation and verification of the limit of detection. Simulation results are used to document performance. CONCLUSION: The proposed procedure appears useful for application in the field of clinical chemistry and promotes a standardized approach for estimating LoDs of clinical chemistry assays.     

Impact of testosterone assay standardization efforts assessed via accuracy-based proficiency testing

BackgroundWe reported observations on analytical performance in testosterone measurements of various methods/assays from the study carried out using accuracy-based proficiency testing (PT) during 2012–2013. In 2016, we re-evaluated analytical performance of testosterone assays using accuracy-based PT to assess effectiveness of CDC efforts toward standardization.MethodsFive single-donor human serum samples from female and male adult donors were analyzed for testosterone by New York State Department of Health-certified clinical laboratories using 16 immunoassays and LC-MS/MS methods. Target values were determined using the CDC reference measurement procedure.ResultsTestosterone targets for the 5 samples were 43.5, 160, 294, 457, and 534 ng/dL. The biases of individual result of the 65 participant laboratories against the target for each sample were calculated. Of participants, 87.7% had ≥4 of the 5 results within the minimum allowable total error limits (± 25.1%), a 14.7% increase from the previous study. The improved PT scores were attributed to better analytical accuracy and precision, and laboratories' selection of more accurate assays/methods.ConclusionsImproved analytical accuracy and precision for testosterone assays were demonstrated over a 3.5-year period after the first CDC-directed accuracy-based proficiency testing. Additional effort is needed to improve accuracy/precision of measurements, especially at low concentrations.