Candidate reference method for determining creatinine in serum: method development and interlaboratory validation

We describe a "high-performance" liquid chromatographic (HPLC) method for accurately determining creatinine in serum. After prechromatographic precipitation of protein, we performed isocratic ion-exchange chromatography with ultraviolet detection (234 nm). Analytical results showed linearity up to 1770 mumol/L, a detection limit of 22 mumol/L, an average analytical recovery of 101%, and a CV ranging from 3% to 11%. We used certified human serum (National Institute of Standards and Technology), and additional lyophilized serum pools also assayed by definitive isotope-dilution mass spectrometry, to validate the accuracy of the HPLC method. In addition, the isocratic HPLC results showed close agreement with those obtained with a step-gradient HPLC method. We also compared the isocratic HPLC method with alkaline picrate and enzymatic methods. Our findings with samples from nonuremic, uremic, and diabetic ketoacidotic patients confirmed the positive bias previously reported with the alkaline picrate method. Interlaboratory transferability of the method was demonstrated with various commercial instruments and analytical columns. We evaluated column stability and possible interference from endogenous or exogenous compounds. On the basis of our analytical findings, we recommend the isocratic HPLC method as a candidate Reference Method for determining creatinine in serum.     

Candidate reference method for total thyroxine in human serum: use of isotope-dilution liquid chromatography-mass spectrometry with electrospray ionization

BACKGROUND: There is a need for a critically evaluated reference method for thyroxine to provide an accuracy base to which routine methods can be traceable. We describe a candidate reference method involving isotope-dilution coupled with liquid chromatography/mass spectrometry. METHODS: An isotopically labeled internal standard, thyroxine-d(5), was added to serum, followed by equilibration, protein precipitation, and ethyl acetate and solid-phase extractions to prepare samples for liquid chromatography-mass spectrometry electrospray ionization (LC/MS-ESI) analysis. For separation, a Zorbax Eclipse XDB-C(18) column was used with a mobile phase consisting of 1 mL/L acetic acid in acetonitrile-water (32:68 by volume) for positive ions and a Zorbax Extend-C(18) column with a mobile phase consisting of 2 mL/L ammonium hydroxide in methanol-water (32:68 by volume) for negative ions. [M + H](+) ions at m/z 778 and 783 for thyroxine and its labeled internal standard were monitored for positive ions and [M - H](-) ions at m/z 776 and 781 for negative ions. Samples of frozen serum pools were prepared and measured in three separate sets. RESULTS: Within-set CVs were 0.2-1.0%. The correlation coefficients of all linear regression lines (measured intensity ratios vs mass ratios) were 0.999-1.000. Positive- and negative-ion measurements agreed with a mean difference of 0.45% at three concentrations (50, 110, and 168 microg/L). The detection limits (at a signal-to-noise ratio of approximately 3 to 5) were 30 and 20 pg for positive and negative ions, respectively. The results from the LC/MS-ESI method were within 1 SD of the composite means from many routine clinical methods, although it appears that the clinical method means may be biased high by 4-5 microg/L across the concentrations. Some routine clinical methods may be biased by up to 20% at low concentrations. CONCLUSIONS: This well-characterized LC/MS-ESI method for total serum thyroxine with a theoretically sound approach, demonstrated good accuracy and precision, and low susceptibility to interferences qualifies as a candidate reference method. Use of this reference method as an accuracy base may reduce the apparent biases in routine methods along with the high interlaboratory imprecision.     

KHC3法测定血清总胆红素的方法评价

[目的] 应用美国临床实验室标准化委员会（NCCLS）的标准化评价方案对KHC3法测定血清总胆红素（TBIL）进行初步评价。[方法] 系统研究KHC3法测定血清总胆红素的精密度、准确度、线性范围及干扰因素，并比较其与重氮法结果的相关性。[结果] KHC，法测定血清总胆红素具有良好的精度（批内，批间，日间及总变异系数均小于5％）和准确度（回收率95．7％～102．2％，平均回收率98．7％）。线性范围：0～513μmol／L．维生素C（VitC）≤0.5g／L、甘油三酯（TG）≤5．0mmoL／L和血红蛋白（Hb）≤1．5g/L时对KHC，法无干扰与重氮法相关性好。[结论]KHC3法测定总胆红素具有准确度高、重复性好，线性范围宽，有较强的抗干扰能力，适用于实验室常规自动化分析。     

Comparison of five routine methods with the candidate reference method for the determination of bilirubin in neonatal serum

Using five routine methods and the candidate reference method of Doumas (Clin. Chem. 31, 1779-1789 (1985)), total bilirubin was determined in 77 neonatal serum samples (concentration range 63-444 mumol/l, average value 227 mumol/l). Four of the routine methods (Jendrassik & Grof's (Biochem. Z. 297, 81-89 (1938)) method, Hertz's (Scand. J. Clin. Lab. Invest. 33, 215-230 (1974)) method, the bilirubinometer procedure, and the method employing 2,5-dichlorophenyldiazonium (Scand. J. Clin. Lab. Invest. 29, Suppl. 126, Abstr. 11. 12. (1972]) gave values that were generally higher than those of the reference method. In contrast, the results from Vink's (Clin. Chem. 34, 67-70 (1988] direct spectrophotometric method differed only negligibly from those of the reference method. The accuracy of Jendrassik & Grof's method, and to a limited extent that of the 2,5-dichlorophenyldiazonium method, can be improved by redetermination of the molar absorption coefficient, or by using a standard containing a matrix of human albumin, with an assigned value determined by the reference method. It was found that Hertz's direct spectrophotometric method can be replaced by that of Vink. The accuracy of the bilirubinometer results could be improved only by using calibrators with assigned values specific for the bilimeter, or by calibration with a serum pool.     

Application of reference methodology: determination of total protein in serum

A study is described on the calibration of the Technicon SMAC for the determination of total protein. Various results of external quality control programmes showed a bias between our value and the mean mentioned in the survey report. A difference of 4 g/l was found to exist between our calibration value and the value mentioned by the manufacturer. The question arises as to which methodology should be chosen in solving the problems described in this article.     

洛阳地区健康人群血清总胆红素参考范围调查

目的了解洛阳地区健康人群血清总胆红素参考值水平。方法采用全自动生化分析仪用钒酸盐氧化法对该地区健康人群2 640例进行血清总胆红素测定,并与操作规程的参考值范围进行对比分析。结果洛阳地区2 640例健康人群总胆红素参考范围分别为:男性8.5～26.6μmol/L,女性8.3～24.9μmol/L,总体8.4～25.7μmol/L。结论洛阳地区健康人群血清总胆红素水平与操作规程参考值范围有较大差异,建议各地区实验室根据不同年龄、性别建立各自的参考值范围。     

Gas-chromatographic determination of cholesterol in serum: candidate reference method

We present a candidate Reference Method for determination of total cholesterol in serum. The method is based on high-resolution capillary gas chromatography, and we took special precautions with respect to weighing, calibration, and chromatographic peak integration. Analytical recovery of added cholesterol was essentially complete (99.99%, SD 0.48%) and reproducibility was excellent (total CV 0.35-0.50%). When cholesterol was determined in a reference serum certified by means of an established candidate Definitive Method, no significant bias could be detected.     

常规方法检测血清总胆红素的正确度评价

目的探讨并比较常规方法检测血清总胆红素(T-Bil)的正确度的评价方法。方法参照CLSI EP15-A2文件,用常规方法测定有证参考物质、国际参考实验室室间比对计划(RELA)样本、卫生部临床检验中心室间质评样本T-Bil水平,用BioRad Unity实验室室间质评数据分析本实验室与对等组和方法组间的偏移及标准差指数(SDI),与参考方法比对等方案评估常规测量T-Bil的正确度。结果 NIST SRM 909b参考物质、2010～2011年RELA样本、2012年卫生部临检中心第1次室间质评样本测定结果均在可接受范围内。2012年1～6月Unity实验室间质评数据结果偏移及SDI指数均满足要求。与参考方法比对结果相关性好(Y=1.129 9X-5.734 3,r=0.999 8),小于300μmol/L时,偏移可接受;大于300μmol/L时,偏移较大(10%～13%),不可接受。用去离子水稀释5倍使其浓度约达到50～100μmol/L后重测,偏移可接受,满足临床需要。结论 "单点浓度"的正确度评价具有局限性,临床实验室应分析全测量范围内的正确度情况,与参考方法比对是实现常规测量正确度评价的有效方法之一。     

BiliChek transcutaneous bilirubin meter overestimates serum bilirubin as measured by the Doumas reference method

ObjectivesTo determine the relationship between BiliChek TcB (Respironics, Marietta GA) and Doumas reference serum or plasma total bilirubin (TSB).Design and methodsPooled samples with values assigned by the Doumas reference method were used to establish the relationship between a local laboratory and reference Doumas TSB. We then established the relationship between TcB and TSB in the 3 months before and after reassignment of calibrator setpoints undertaken to match the local laboratory to Doumas reference bilirubin values.ResultsBefore calibrator setpoint reassignment TSB as measured in our laboratory overestimated Doumas reference bilirubin. After calibrator adjustment laboratory TSB was within 1.7–6.8 µmol/L (0.1–0.4 mg/dL) of Doumas reference values. Mean bias between BiliChek TcB and TSB was 42.8 ± 22.2 µmol/L (2.5 ± 1.3 mg/dL) (n = 94) before and 49.6 ± 22.2 µmol/L (2.9 ± 1.3 mg/dL) (n = 115) after calibration adjustment.ConclusionsBiliChek TcB significantly overestimates TSB as measured by the Doumas reference method.