Description of a generally applicable model for the evaluation of uncertainty of measurement in clinical chemistry.

There is a growing pressure on clinical chemistry laboratories to conform to quality standards that require the evaluation and expression of the uncertainty of results of measurement. Nevertheless, there is some reluctance to accept the uncertainty concept in the analytical community due to difficulty in evaluating uncertainty in practice. For example, often the uncertainty of some uncertainty components is not known very well in clinical chemistry measurements, such as those associated with matrix effects or with the values of the calibrators. Moreover, it is not clear how to interpret uncertainty in relation to diagnostic criteria, reference ranges and other decision limits in clinical chemistry practice. Hence, the value of reporting the uncertainty of the measurement result is not obvious. In this paper it is suggested a relatively simple, logical procedure for evaluating measurement uncertainty based on the principles in the Guide for the Expression of Uncertainty of Measurement (GUM). The measurement process is partitioned into elements that are well known to the analyst, namely sampling, calibration, and analysis. The corresponding model function expresses the result of a measurement as the value obtained by the analytical procedure multiplied by the correction factors for sampling bias, for bias caused by the calibrators, and for other types of bias. Under normal conditions, when the measurement procedure is validated and corrected for all known bias, the expected value of each correction factor is one. The uncertainty that remains with regard to sampling, manufacturing of calibrators and other types of bias is combined with the analytical imprecision to yield a combined uncertainty of a result of measurement. The advantages of this approach are: (i) Data from the method validation, internal quality control and from participation in external quality control schemes can be used as input in the uncertainty evaluation process. (ii) The partition of the measurement into well-defined tasks highlights the different responsibilities of the clinical chemistry laboratory and of the manufacturer of reagents and calibrators. (iii) The approach can be used to harmonize the uncertainty evaluation process, which is particularly relevant for laboratories seeking accreditation under ISO 17025. The application of the proposed model is demonstrated by evaluating the uncertainty of a result of a measurement of prolactin in human serum. In the example it is shown how to treat the uncertainty associated with a calibrator supplied with a commercial analytical kit, and how to evaluate the uncertainty associated with matrix effects.     

Does the uncertainty of commonly performed glucose measurements allow identification of individuals at high risk for diabetes?

Revised recommendations for diagnosis of diabetes introduce the intermediary risk group of impaired fasting glucose (IFG), defined as individuals with a fasting blood-glucose concentration between 5.6 and 6.0 mmol/l. We apply the concept of uncertainty to identifiable steps of sampling and measuring blood-glucose. Since many instruments in primary health care measure plasma-glucose and report results as blood-glucose and vice versa, factors affecting the transformation are also considered. The study identifies the measurement procedure as the major source of uncertainty, closely followed by preanalytical sources. The estimated uncertainties indicate that the presently available procedures do not allow identification of IFG by a single investigation. The approach to establish an uncertainty budget can be used to evaluate the clinical usefulness of measurements.     

Non-homogeneous separation of triglycerides, gamma-glutamyltransferase, C-reactive protein and lactate dehydrogenase after centrifugation of lithium-heparin tubes

BACKGROUND: Clinical chemistry testing is influenced by a variety of preanalytical variables, including sample preparation. The presence of a diluted plasma layer at the top of primary tubes containing plasma citrate has recently been reported. However, no indication is available so far on the potential non-homogeneous distribution of clinical chemistry analytes during centrifugation of primary tubes containing lithium-heparin as an additive. METHODS: A total of 40 lithium-heparin plasma samples were collected from volunteers and immediately centrifuged. An aliquot was obtained from the upper 0.4 mL of plasma (upper aliquot), 1.0 mL of plasma was discarded, and a second aliquot (lower aliquot) was obtained from the remaining plasma. The concentrations of alanine aminotransferase, albumin, alkaline phosphatase, amylase, amylase pancreatic, aspartate aminotransferase, direct bilirubin, total bilirubin, blood urea nitrogen, calcium, chloride, cholesterol, C-reactive protein (CRP), creatinine, creatine kinase, gamma-glutamyltransferase (GGT), glucose, high-density lipoprotein-cholesterol, iron, lactate dehydrogenase (LDH), magnesium, phosphate, potassium, total protein, sodium, triglycerides and uric acid were assayed on a Roche/Hitachi Modular System P according to the manufacturer's specifications and using proprietary reagents. Sodium, chloride and potassium were measured on a Roche/Hitachi Modular System using indirect ion-selective electrode methods. RESULTS: We observed a statistically significant difference between the upper and lower aliquots for CRP (3.88+/-0.67 vs. 3.94+/-0.68 mg/L; p=0.025), GGT (32.1+/-8.0 vs. 31.8+/-8.0 U/L; p=0.013), LDH (395+/-19 vs. 386+/-20 U/L; p=0.010) and triglycerides (1.29+/-0.09 vs. 1.27+/-0.09 mmol/L; p=0.001); results for the other analytes were not significantly different. In no case did the mean percentage bias recorded between aliquots exceed the current analytical quality specifications for desirable bias. CONCLUSIONS: The results of our investigation show that plasma layer stratification might occur in primary lithium-heparin tubes for a limited number of routine clinical chemistry tests, introducing a statistically significant bias in the measurement of GGT, LDH, triglycerides and CRP in the upper vs. the bottom section. When delayed testing is necessary for these parameters, we suggest that plasma should be separated after centrifugation and appropriately mixed before delayed/repeated analysis or aliquoting.     

Optimization of preanalytical conditions and analysis of plasma glucose. 1. Impact of the new WHO and ADA recommendations on diagnosis of diabetes mellitus

The new diagnostic criteria for type 2 diabetes from the American Diabetes Association (ADA) and World Health Organization (WHO) recommend measurements on plasma and a lowering of the glucose threshold for diabetes by 0.8 mmol/L. This narrows the distance between the upper end of the reference limit and the discriminatory level to a degree where analytical quality becomes critical. The quality demands for the preanalytical and analytical phase and their consequences on diagnostic performance have to be established in the new technical system, measuring in plasma rather than in capillary whole blood. Because of the instability of glucose in blood samples it is necessary to clarify the influence of different preanalytical and analytical factors on the number of false-positive and false-negative classifications. Thus the aim of the present study was to find optimal conditions for sampling, additives, storage, transport and analysis of plasma glucose combining feasibility with an analytical bias close to zero and a within-imprecision around 1%. We have documented the analytical performance of the method itself and its traceability to an international standard. The preanalytical conditions, such as influence of antiglycolytic agent NaF, conditions for plasma separation, storage temperature and storage time before and after plasma separation were investigated. In conclusion, we recommend that blood should be drawn in tubes containing heparin and NaF and kept on ice water for not more than 1 h until centrifugation at minimum 1000 x g for 10 min. The plasma is then stable for at least 48 h at room temperature.     

Severe retinopathy in type 1 diabetic patients is not related to the level of plasma homocysteine

The new diagnostic criteria for type 2 diabetes from the American Diabetes Association (ADA) and World Health Organization (WHO) recommend measurements on plasma and a lowering of the glucose threshold for diabetes by 0.8mmol/L. This narrows the distance between the upper end of the reference limit and the discriminatory level to a degree where analytical quality becomes critical. The quality demands for the preanalytical and analytical phase and their consequences on diagnostic performance have to be established in the new technical system, measuring in plasma rather than in capillary whole blood. Because of the instability of glucosein blood samples it is necessary to clarify the influence of different preanalytical and analytical factors on the number of false-positive and false-negative classifications. Thus the aim of the present study was to find optimal conditions for sampling, additives, storage, transport and analysis of plasma glucose combining feasibility with an analytical bias close to zero and a within-imprecision around 1%. We have documented the analytical performance of the method itself and its traceability to an international standard. The preanalytical conditions, such as influence of antiglycolytic agent NaF, conditions for plasma separation, storage temperature and storage time before and after plasma separation were investigated. In conclusion, we recommend that blood should be drawn in tubes containing heparin and NaF and kept on ice water for not more than 1h until centrifugation at minimum 1000 g for 10min. The plasma is then stable for at least 48h at room temperature.     

基于电感耦合等离子体质谱法血清钙离子检测候选参考方法的建立

目的基于电感耦合等离子体质谱法(ICP-MS),建立一种检测血清钙离子的候选参考方法。方法方法学建立.将从医院收集到的血清标本以0.3%硝酸溶液直接稀释100倍,在血清标本基质溶液中添加不同浓度钙标准溶液,配制含血清基质的标准品溶液。以锗(Ge)为内标,采用标准加入法,计算血清钙离子浓度。对所建立的候选参考方法进行线性、精密度、正确度的性能评估和方法比对。结果血清钙离子浓度在0.000~20.400 mmol/L内(稀释后浓度为0.000~0.204 mmol/L)线性良好(R2>0.9999);批内不精密度为0.22%~0.47%,批间不精密度为0.64%~0.77%,总不精密度为0.98%~1.09%;检测3个浓度SRM 956d,结果均在证书要求的不确定度范围内,相对偏移分别为-0.16%,0.04%,0.23%;该方法参加2017年参考实验室外部质量评价计划(RELA),比对通过。与检验医学溯源联合委员会(JCTLM)所列参考方法进行比较,结果一致性良好。本研究所建立的候选参考方法与临床常规电极方法进行比较,具有良好的相关性。结论成功建立血清钙离子检测候选参考方法,线性范围宽、具有良好的精密度与准确度。     

Comparison and validation of point of care lactate meters as a replacement for fetal pH measurement

Objective

To validate a point of care lactate device to replace fetal pH measurement.

Study design and methods

Cord blood samples drawn immediately following delivery were tested on the Nova Lactate Plus and ARKRAY Lactate Pro, the Corometrics 220 pH System, and the Vitros chemistry analyzer (used as lactate reference).

Results

Nova demonstrated a constant positive bias relative to the lactate reference method; while the Lactate Pro correlated well with the reference method up to 6 mmol/L. Receiver operating characteristic (ROC) curve analysis showed optimal sensitivity and specificity for predicting pH < 7.20 at lactate values of 6.8 mmol/L for the Nova and 4.8 mmol/L for the Lactate Pro.

Conclusion

Using Lactate Pro the best cut-off for predicting pH ≤ 7.20 was 4.8 mM; which coincides with current clinical cut-offs. Thus any lactate device that correlates well with the laboratory reference method can be used with a clinical cut-off of 4.8 mmol/L.     

干化学法与湿化学法检测尿素氮和肌酐结果相关性及偏倚分析

目的：比较干化学法与湿化学法检测尿素氮（BUN）、肌酐（CREA）结果，分析两者的相关性并进行偏倚评估。方法利用VITROS 5600干化学分析仪和Olympus AU5421全自动生化分析仪检测40例血清BUN、CREA，对结果进行统计分析。结果BUN相关回归方程Y=1.0023X-0.0983，r=0.9987。当BUN浓度为5 mmol/L，15 mmol/L，25 mmol/L时，干化学法的相对偏倚分别为1.72%，0.045%，0.018%。CREA相关回归方程Y=0.9764X-0.7654，r=0.9927。结论干化学法和湿化学法检测结果高度相关，BUN、CREA检测结果差异无统计学意义，应定期进行结果比对和校正，使结果具有可比性。     

Selection, validation, standardization, and performance of a designated comparison method for HDL-cholesterol for use in the cholesterol reference method laboratory network.

BACKGROUND: Accurate and precise HDL-cholesterol (HDL-C) measurements are essential for effective application of National Cholesterol Education Program treatment guidelines. The Cholesterol Reference Method Laboratory Network (CRMLN) assists manufacturers of in vitro diagnostic products to establish traceability to the accuracy base. CRMLN sought to implement a designated comparison method (DCM) that overcomes the impracticalities of the expensive and labor-intensive reference method for HDL-C. METHODS: CRMLN evaluated candidate DCMs and selected one that uses 50-kDa dextran sulfate with magnesium ions as the precipitation reagent followed by measurement of cholesterol by the CDC reference method. After validating the method, we transferred it to all CRMLN laboratories and successfully standardized it using CDC frozen serum reference materials. CRMLN laboratories participate in monthly performance evaluations. RESULTS: CRMLN laboratories were able to meet a precision goal, as indicated by SD, of /=1.09 mmol/L (42 mg/dL) 95.6% of the time. CRMLN is working to further improve its performance by implementing a bias criterion of 0.03 mmol/L (1 mg/dL) for all HDL-C concentrations. CONCLUSIONS: CRMLN selected, validated, standardized, and implemented a DCM for HDL-C that is accurate, robust, transferable, and practical. The DCM is being used to assist manufacturers in calibrating their products so that ultimately, clinical laboratories using the products will more accurately measure HDL-C.     

国产循环酶法检测同型半胱氨酸试剂盒的分析性能评价

目的对某国产循环酶法同型半胱氨酸试剂盒进行性能评价。方法依据CLSI-EP相关文件,在AU2700生化仪上对该试剂盒的精密度、线性范围、可报告范围、生物参考区间验证、干扰试验等方面进行性能评价;并与西门子化学发光法进行比对分析,计算相关系数和直线回归方程,根据回归方程计算同型半胱氨酸在医学决定水平处的预期偏倚和预期偏倚的95%可信区间并判断偏倚是否可以接受。结果批内精密度(CV)为0.94%、1.41%,批间CV为3.00%、2.60%;线性范围5.0~50.0μmol/L,最大稀释倍数为20,临床可报告范围5.0~1 000.0μmol/L;血红蛋白≤5g/L、三酰甘油≤7.8mmol/L、抗坏血酸≤1.7mmol/L、胆红素≤0.68mmol/L,对试验无明显干扰;两种分析仪结果间均呈良好的线性关系,其相关系数r均大于0.975,在医学决定水平处的系统偏倚临床可以接受。结论该国产同型半胱氨酸测定试剂盒在AU2700全自动生化仪上的分析性能良好,可应用于临床检测。     

How good are clinical chemistry laboratories at analysing ethylene glycol?

The results of an external proficiency test of clinical chemistry laboratories in Sweden when the target analyte was ethylene glycol (EG) are presented. Specimens of plasma were spiked with EG (10% w/v) to give assigned concentrations ranging from 5 to 50 mmol/L. Over a period of 6 years, two control specimens of plasma were sent for analysis on 21 occasions to between 14 and 20 participating laboratories as a declared proficiency trial. The analytical precision between and within laboratories was determined by spiking the plasma specimens with the same concentration of EG so that the results reported back could be considered a duplicate determination. On one occasion propylene glycol (PG) was substituted for EG without informing the participants. The standard deviation (SD) within laboratories expressed as the coefficient of variation (CV) was 4.5% compared with 11.4% between laboratories. Results reported by laboratories using gas chromatography (GC) were in good agreement with those when an enzymatic method was used. The between-laboratory SD increased with concentration of EG in the specimen and at a mean concentration of 18 mmol/L, the pooled SD was 4.11 mmol/L (CV = 23%). Four laboratories reported finding EG in plasma when PG was the diol present; three laboratories used an enzymatic method and one used GC. Clinical laboratories that provide a toxicology service should regularly participate in external quality assurance schemes that include low-molecular-weight alcohols such as EG. Efforts should be made to standardize the analytical methods used for toxicological analysis.     

Nuclear specific thyroxine binding in human mononuclear blood cells

The results of an external proficiency test of clinical chemistry laboratories in Sweden when the target analyte was ethylene glycol (EG) are presented. Specimens of plasma were spiked with EG (10% w/v) to give assigned concentrations ranging from 5 to 50?mmol/L. Over a period of 6 years, two control specimens of plasma were sent for analysis on 21 occasions to between 14 and 20 participating laboratories as a declared proficiency trial. The analytical precision between and within laboratories was determined by spiking the plasma specimens with the same concentration of EG so that the results reported back could be considered a duplicate determination. On one occasion propylene glycol (PG) was substituted for EG without informing the participants. The standard deviation (SD) within laboratories expressed as the coefficient of variation (CV) was 4.5% compared with 11.4% between laboratories. Results reported by laboratories using gas chromatography (GC) were in good agreement with those when an enzymatic method was used. The between-laboratory SD increased with concentration of EG in the specimen and at a mean concentration of 18?mmol/L, the pooled SD was 4.11?mmol/L (CV=23%). Four laboratories reported finding EG in plasma when PG was the diol present; three laboratories used an enzymatic method and one used GC. Clinical laboratories that provide a toxicology service should regularly participate in external quality assurance schemes that include low-molecular-weight alcohols such as EG. Efforts should be made to standardize the analytical methods used for toxicological analysis.     

用患者样本进行方法学对比及在两种葡萄糖测定法中的应用

目的 用患者样本分析葡萄糖氧化酶-氧电极法(简称电极法)和己糖激酶(HK)法测定血清葡萄糖(GLU)时的偏倚。方法 依据美国临床实验室标准化委员舍(NCCLS)EP9—A文件，每天取患者样本8份，分别用两种方法测定血清葡萄糖含量，共测定5d，记录检验结果，去除离群点，计算线性方程和相关系数，并进行偏倚估计。结果 在进行患者葡萄糖测定时，电极法(Y)和HK法(X)测定结果的回归方程为：Y＝0．9857X 0．08967，r^2=0．9992；电极法和HK法测定结果的预期相对偏倚在GLU＝15mmo1／L时为0．80％，GLU＝9mmo1／L时为0．44％，GLU＝6mmo1／L时为0．00％，GLU＝3mmo1／L时为1．67％。结论 电极法和HK法测定血清葡萄糖时，测定结果在低浓度时偏倚较大，在中、高浓度时偏倚较小，两法间有良好的相关性。     

氰化高铁血红蛋白国家一级标准物质的研制

目的 研制氰化高铁血红蛋白(HiCN)国家一级标准物质,用作血红蛋白测定结果溯源的标准.方法 参照国际血液学标准化委员会(ICSH)的要求,制备HiCN标准物质;按照ISOGuide 35的要求,评价标准物质的均匀性和稳定性,在二者合乎要求的基础上,由多个实验室使用溯源至美国国家标准技术研究所(NIST)标准滤光片的分光光度计为HiCN标准物质定值;为验证定值结果的可靠性,使用定值仪器测定国际标准物质,将测定结果与WHO参考实验室的定值进行比较,此外,对所研制标准物质和国际标准物质的扫描图形进行了比较.结果 HiCN标准物质均匀性的不确定度为0.000 4 g/L,变异系数(CU)为0.09%;长期稳定性的不确定度为0.000 6 g/L;HiCN标准物质的定值为0.6159 g/L,不确定度为0.000 4 g/L;当扩展因子取2时,标准物质的扩展不确定度为0.001 8 g/L;定值仪器对国际标准物质的测定结果与国际标准物质定值的相对偏差为0.08%.结论 HiCN标准物质均匀性和稳定性良好,定值方法准确、可靠.     

A model for the effect of bias for cholesterol on the population at risk

The location of the Reference Value for an analyte within the population distribution affects the magnitude of error due to methodological bias. Using the gaussian distribution, we evaluated the effects of systematic and proportional biases of the method (positive and negative), mean value, and standard deviation on the magnitude of error. We chose four Reference Values for cholesterol as a model. For a population with a mean of 2.0 and SD of 0.36 g of cholesterol per liter, a 3% positive proportional bias causes sixfold more error at the 50th percentile than at the 97.5th. In general, the error for a given bias (proportional or systematic) is greater for a Reference Value within the body than at the tails of the distribution. Further, the magnitude of the error varies as a function of the mean and standard deviation of the population.     

速率法检测献血者丙氨酸转氨酶的测量不确定度评定

目的通过丙氨酸转氨酶（ALT）测量不确定度的评定,探讨血站实验室建立测量不确定度的评定方法和程序。方法依据（（JJFl059-1999测量不确定度评定与表示》和《CNAS-GL05测量不确定度要求的实施指南》对ALT速率法的测量不确定度进行评定,明确测量不确定度分量来源,采用不确定度A类和B类评定方法评定各不确定度分量,计算合成不确定度与扩展不确定度。结果献血者血浆ALT浓度为52．5U／L时,其扩展不确定度为U=3．31U／L（包含因子k=2,置信区间P=95％）。结论本实验室所建立的测量不确定度评定方法可分析不同因素对测量结果的影响程度,有助于实验室提高检测质量。     

Electrophoretic separation of high-density lipoprotein choelsterol evaluated and compared with the modified lipid research clinic procedure.

We evaluated a new agarose-gel-electrophoretic procedure (Corning) (I) for separating and quantitating of high-density lipoprotein cholesterol (HDLC), comparing it with the modified Lipid Research Clinics (LRC) procedure (heparin 183 kilounits/L, MnCl2 92 mmol/L) (II). Method I was insensitive to an HDLC concentration of 50 mg/L, but gave a linear dose-response curve between 130 and 1200 mg/L. Method II is sensitive to 50 mg/L and linear from 50--1200 mg/L. The within-plate CV for the Corning method varied from 26.2% for an HDLC of 168 mg/L to 6.8% for 580 mg/L. Within-day between-plate CV for the Corning method ranged from 22.1% at 155 mg/L to 8.0% at 651 mg/L, compared to 3.0 and 0.8% for the modified LRC procedure. Between-day CV for method I was 20, 12.6, 4.3, and 3.5% for HDLC concentrations of 175, 435, 542, and 678 mg/L, respectively; for method II it was 14, 5, 3.5, and 2.6%, respectively. Analysis of HDLC in 100 patients by both procedures showed mean HDLC values to be significantly lower (mean + SD, 27.8 +/- 1.7 mg/L; p less than 0.001) by method I. In 46 patients with HDLC less than 450 mg/L, this difference was accentuated (mean + SD = 40.5 +/- 2.6 mg/L) and clinically significant. Electrophoretic methods offer a promising further alternative method for HDLC separation and quantitation, but the negative bias, present limited sensitivity, and lack of precision at less than 450 mg/L indicate that they are not yet optimal for routine clinical use for patients with values less than 450 mg/L.     

Facilitated determination of ionized calcium

Using a calcium-containing heparin preparation for anticoagulation, we determined [Ca2+], the mean concentration of ionized calcium, in whole blood of 120 healthy blood-donors to be 1.23 (SD 0.04) mmol/L. Similarly, for 50 intensive-care patients selected without conscious bias, the correlation between [Ca2+] in serum (mean 1.15, SD 0.10 mmol/L) and in whole-blood samples anticoagulated with the same heparin preparation (mean 1.15, SD 0.09 mmol/L) was very good (r = 0.95). Storing samples anaerobically on ice for as long as 2 h did not alter whole-blood [Ca2+]. On the other hand, various concentrations of calcium-free heparin preparations all induced a significant decrease in measured [Ca2+]. By using whole-blood samples, rather than plasma or serum, for [Ca2+] determination with a calcium-selective electrode, repetitive measurements can be made with simple handling procedures, facilitating rapid implementation of appropriate therapeutic measures for critically ill patients.     

Enzymic assay for oxalate in unprocessed urine, as adapted for a centrifugal analyzer

In this automated modification of the oxalate decarboxylase method, oxalate can be measured (12 per hour) in acidified but otherwise unprocessed urine. Standard curves are linear up to at least 2.5 mmol/L. When 0.50 mmol of oxalate was added per liter to samples of 18 patients' urines, a mean analytical recovery of 98.5% (SD 3.6%) was obtained. Within-series CVs were 3.4 and 1.0%, between-series CVs 7.3 and 2.7% (n = 15) for oxalate concentrations of 0.31 and 0.61 mmol/L. The lower limit of detection is 25 mumol/L. Concentrations measured with this "direct" method correlated well (r = 0.95) with those measured after precipitation with calcium and ethanol and resolubilization in dilute sulfuric acid. For 17 healthy volunteers the mean urinary excretion of oxalate was 0.37 (SD 0.14) mmol/24 h.     

血清钾测定的国际比对

目的：通过参加国际环形比对试验,验证该实验室的分析检测能力。方法将钴元素作为内标加入血清样品,1％硝酸将血清准确稀释100倍,选择和优化电感耦合等离子质谱（ICP-MS）分析条件后,测定国际比对样本A和B。美国国家标准与技术研究机构（NIST）血清标准物质SRM909bⅠ用来评价方法的准确度。比对样本测定3批,每批测定4次,以考察分析的批内和批间精密度及总不确定度。结果 SRM909bⅠ测定均值在“靶值±不确定度[（3．424±0．025）mmol/L]”范围内。比对样本A、B两水平的测定结果分别为（4．08±0．033）mmol/L,（2．86±0．025）mmol/L,与平均值相比偏倚分别为0．87％和0,在规定的“等效限”±2％范围内。A、B两水平的样本测定总变异系数为0．18％～0．23％（狀＝12）。结论比对工作顺利完成,在技术水平上与国外同类实验室水平相当,进一步验证血清钾电感耦合等离子体质谱钴内标法准确、可靠。