Comparison between the triglycerides determination by HPLC in China and reference procedure used in the isotope dilution mass spectrometry CDC CRMLN / 中华检验医学杂志

Objective To evaluate the comparability and consistency of two kinds of triglycerides reference methods, one of which is the isotope dilution liquid chromatography-mass spectrometry (LC/MS) in the Cholesterol Reference Method Laboratory Network (CRMLN), the other isthehigh-performance liquid chromatography (HPLC) method for triglyceride detection in China. Methods 52 fresh frozen sera with triglycerides levels among 0.45-4.52 mmol/L were determined by LC/MS and HPLC. After evaluation the precision and accuracy of the two methods,a series of analyses were conducted including plotting to scatter plots and deviation graphs, testing outliers, selecting the best fitting regression models and calculating the regression equations and parameters, and so on. The expected deviation at the level of medical decision of triglycerides and the 95％confidence range were statistically analyzed.Results For HPLC method, the CV of instrument measurement was 0.29％(0％-1.16％), the total CV of samples measurement was 0.54％(0.04％-1.28％), and the average bias of the reference materials was 0.22％(-0.43％-0.68％). ForLC/MSmethod,the CV of instrument measurement was 0.55％(0％-1.68％),the total CV of samples measurement was 0.79％(0％-1.93％), and the average bias of the NIST reference materials was 0.09％(-0.73％-1.29％). No outlier was found from the scatter plots and the statistical analysis and the linear regression was fitted to analyze the results of the two methods. The linear regression parameters of two methods for 52 fresh frozen human sera were as follows:the slope was 0.9988,the standard error of slope was 0.0035, the intercept was 0.0037mmol/L, the standard error of intercept was 0.0030 mmol/L, the standard error of Y-estimate was 0.0236 mmol/L,and the correlation coefficient was 0.9997. Compared with the LC/MS method,the absolute deviation of fresh sera by HPLC method was-0.001 mmol/L (-0.070-0.056 mmol/L), with a relative deviation of 0.13％ (-2.01-2.83％). T-test showed no statistically significant difference between the two methods. The expected deviations at the triglycerides medicine decision level were within the 95％confidence range,and the expected deviations were far less than the allowable error. Conclusions The HPLC method of triglyceridesdetetion has good consistency and comparability with LC/MS method as one of the reference methods of CRMLN. Because of the advantages of HPLC method such as low cost, simplicity,less technical need,and better precision,HPLC method is expected to play an important role in the process of standardization and traceability of serum triglycerides.

Preparation of cholesterol and glycerol reference materials in cyclodextrin aqueous solution / 中华检验医学杂志

Objective Preparation of aqueous reference materials for cholesterol and glycerol.Methods Study on reference materials.The certified reference materials GBW09203b and GBW09149 were weighed accurately and dissolved into 20% of methyl cyclodextrin aqueous solution to prepare six kinds of candidate reference materials of cholesterol and glycerol according to the concentration.The materials were tested for homogeneity and stability using routine methods.The reference methods of isotope dilution liquid chromatography tandem mass spectrometry (ID-LC/MS/MS) were used to determine the concentration of cholesterol and glycerol to evaluate the accuracy of the certified values.Meanwhile, the blank verification test was carried out.The expanded uncertainty was the combination of standard uncertainty of measurement, unhomogeneity and instability.Results It showed that the six candidate reference materials were homogeneous and stable for at least 1 year at-70 ℃ and-20 ℃.The certified values (reference value ± expanded uncertainty,mmol/L) were as follows,for cholesterol:0.65±0.01,1.31 ±0.01,2.57±0.02,5.21±0.06,7.71±0.08,10.24±0.06;for glycerol:0.29±0.01,0.58±0.01,1.22±0.02,2.24±0.02,3.46±0.04,4.52 ±0.04.The results of reference methods were consistent with the certified values.Blank validation tests showed that the concentration of the analytes would not be affected by the reagent and the blank matrix.Conclusions Certified reference materials for cholesterol and glycerol in aqueous solution have been prepared successfully.These materials are homogeneous and stable, and the certified values are reliable.Therefore the materials have been approved to be the Certificate Reference Materials of GBW 09823, GBW 09824, GBW 09825, GBW09826, GBW09827 and GBW 09828.

Comparison and bias estimation of isotope dilution mass spectrometry and HPLC for cholesterol:methodological comparison of two internationally accepted reference methods listed in JCTLM / 中华检验医学杂志

Objective To evaluate the consistency and comparability of two kinds of cholesterol reference methods listed in the Joint Committee for Traceability in Laboratory Medicine(JCTLM).Methods 52 fresh frozen sera with cholesterol concentrations among 3 -10 mmol/L were tested by isotope dilution liquid chromatography tandem mass spectrometry (ID-LC/MS/MS)and high performance liquid chromatography(HPLC).The precision and accuracy of the two methods were calculated,and then a series of analysis were conducted including plotting scatter plots and deviation graphs,testing outliers,selecting the best fitting regression models and calculating the regression equations and parameters,and so on.The results of the two methods were statistically analyzed to estimate the expected deviation at the level of medical decision of cholesterol and the 95% confidence range.Results For HPLC method,the CV of instrument measurement was 0.22%(0 -0.49%),the total CV of samples measurement was 0.36%(0.02% -0.83%),and the average bias of the reference materials was 0.37%(-1.31%-0.14%).For ID-LC/MS/MS method,the CV of instrument measurement was 0.50%(0 -1.51%),the total CV of samples measurement was 0.55%(0.03%-1.17%),and the average bias of the reference materials was 0.24%(-0.53%-0.14%).No outliers were found from the scatter plots and the statistical analysis and the linear regression were fitted to analyze the results of the two methods.The linear regression parameters of two methods for 52 fresh frozen human sera were as follows:the slope was 0.989 5,the standard error of slope was 0.003 4,the intercept was 0.063 4 mmol/L,the standard error of intercept was 0.019 9 mmol/L,the standard error of Y-estimate was 0.034 8 mmol/L,and the correlation coefficient was 0.999 7.Compared with the ID-LC/MS/MS method,the absolute deviation of fresh sera by HPLC method was 0.000 mmol/L (-0.083-0.076 mmol/L),with a relative deviation of 0.07%(-1.22-1.24%).T-test results showed no statistically significant difference between the two methods.The expected deviations at the cholesterol medicine decision level were within the range 95% confidence range,and the expected deviations were far less than the allowable error.Conclusions The HPLC method of cholesterol has good consistency and comparability with ID/MS method using the primary measurement principle.Because of more advantages of HPLC method such as less cost,more simple,requirement,and better precision,HPLC method is expected to play an important role in the process of standardization and traceability of serum cholesterol.

Preparation of reference materials of total thyroxine / 中华检验医学杂志

Objective To prepare the serum reference materials for total thyroxine .Methods Individual blood samples were collected from 13 healthy donors (7 males and 6 females) aged from 20 to 50 years old, and the sera were separated and mixed into 4 serum pools according to the concentration of thyroxine.The materials were tested for homogeneity and stability using routine methods .The method of isotope dilution liquid chromatography tandem mass spectrometry ( ID-LC/MS/MS) was used to determine the concentration of thyroxine .The candidate reference materials were also measured by four conventional methods to analyze the commutability of the materials .Results It showed that the four candidate reference materials were homogeneous and commutable in four conventional methods and they were tested to be stable for at least 1 year at -70 ℃using the isochronous stability study .The certified values ( reference value ± expanded uncertainty ,nmol/L) were:75.9 ±1.8,105.3 ±2.2,114.7 ±2.1 and 187.4 ±2.9.Conclusions Certified reference materials for serum thyroxine have been prepared .These materials have been approved to be the Certificate Reference Materials of GBW 09127,GBW 09128,GBW 09129 and GBW 09130.

Trueness evaluation of 13 routine measurements for serum uric acid / 中华检验医学杂志

Objective To evaluate the trueness of 13 routine measurements for serum uric acid and the role of reference method in improving harmonization and trueness among routine measurement systems. Methods The research is related to the reagent evaluation.Usingisotope dilution liquid chromatography tandem mass spectrometry ( ID-LC/MS/MS) method as the comparison method, Wako, Sekisui, DiaSys, Maker,Dirui,Leadman,BSBE,Biosina,Mindray,MedicalSystem,LongMarch,and Kehua 13 kinds of uric acid kits were chosen as the evaluation methods with Hitachi 7170A as the analyzer.serum uric acid in 40 fresh frozen serawere collected from clinical laboratory of Beijing hospital in 2014,coveringboth physiological and pathological status ( 80 -940 μmol/L ) .19 kinds of prepared materials and the 40 fronzen sera were measuredby comparison method and evaluation methods and linear regression analysis was made for the results.The performance of evaluation methods was revealed and recalibration was performed on every evaluation methodby the linear regression equation.The variation of percent bias(%) of the uric acid values in 19 preparation materials was compared.Results All test methods demonstrated good precision ( CV0.998, P<0.01) with the comparison method when measuring uric acid values in 40 fresh frozen sera The meanpercent bias was 0.17% ( -3.06% -7.31%).After recalibration, 4 of 19 samples with no matrix effect values percent bias reduced and met the demands of quality ( <4.8%) induced from biological variation.Conclusion All test methods demonstrated good trueness and their calibration traceability was verified.Recalibration using reference method or standard reference materials contributes to harmonization among methods.

Quality assessment of seven kinds of sero-enzyme tests in China / 中华检验医学杂志

Objective To evaluate the analytical quality of different analytical systems in measuring seven kinds of sero-enzymes consisting of Alanine Aminotransferase(ALT), Aspartate Aminotransferase (AST),γ-Glutamyltransferase(GGT), Lactate Dehydrogenase(LDH), Creatine Kinase(CK), α-Amylase (AMY) and Alkaline Phosphatase(ALP).Methods Data from 2013 routine chemistry external quality assessment (EQA) and Enzymes Trueness Verification(ETV) were collected.1 450 and 165 participating laboratories were selected respectively for investigation.Analytical systems of participating laboratories were classified into 6 kinds,i.e.imported matching system(AI), domestic matching system(AH), systems consisting of imported reagents and corresponding calibrators(BI), systems consisting of domestic reagents and corresponding calibrators ( BH ) , unmatched systems using imported calibrators ( CI ) and unmatched systems using domestic calibrators ( CH ) .Total error, bias and coefficient of variation within laboratories ( CVI) were calculated from the data of 2013EQA and ETV The proportion of laboratories meeting the desirable and the optimal criteria derived from biology variation were analyzedby EXCEL2010 with coincidence rate (CR) above 85% as evaluation criterion.Results The AI and CI occupied more than 70%among six systems, CH occupied approximate 15% and the other systems were less than 10%.The range of the average of ETV′s total errors , EQA′s total errors, absolute value of bias and CVI of seven kinds of sero-enzymes were 6.2%-27.8%, 4.0%-7.0%, 4.2%-25.1% and 3.6%-4.6% respectively. Accuracy, bias and within-laboratory imprecision were judged by CR of ETV′s total errors, ETV′s bias, CVI and EQA′s total errors respectively and comparability between different systems was evaluated.It turned out that the results of analytical systems of enzymes except ALP were comparable, the accuracy of systems of enzymes except AMY, ALP and GGT, LDH of AI, the within-laboratory imprecision of enzymes except LDH, AMY, ALP and AST of AI, CH could meet the desirable criteria.The bias of all systems of seven kinds of sero-enzymes were undesirable.Conclusions The analytical quality of routine testing of seven kinds of sero-enzymes could fulfill the clinical requirement generally in China.

The commutability of reference materials for serum glucose measurements / 中华检验医学杂志

Objective To evaluate the commutability of certified reference materials, external quality assessment program materials and calibrators for serum glucose measurements which were performed in 24 routine measurement procedures.Methods 35 fresh patient specimens and some reference materials were analyzed by isotope dilution liquid chromatography tandem mass spectrometry ( as the comparative method) and 24 routine measurement procedures (as the evaluated methods).The relationships between the results from the evaluated method and the comparative methods were evaluated to identify the commutability.Results It showed that 5 certified reference materials, 2 trueness verification materials, and 5 calibrators were commutable in all 24 routine measurement procedures.The other samples were displayed the presence of commutability issue in different degrees.Conclusion It is important to pay more attention to the problems brought by commutability of reference materials in clinical laboratory.

Pay more attention on the clinical value of hemoglobin A1c in the management of diabetes care / 中华检验医学杂志

Diabetes is a worldwide epidemic disease in the twenty-first century.The most danger of diabetes is its complications which are the major cause of morbidity and mortality in diabetic patients.The development and progression of diabetes complications are directly associated with blood glucose levels,Hemoglobin A1c is not only the gold standard for the long-term blood glucose control of diabetic patients,but also the World Health Organization (WHO) recommended diagnosis of diabetes.Thus,a question has been raised:how to improve the comparability of hemoglobin A1c results meet clinical needs? At least four points should be taken in determining hemoglobin A1c:(1)Understand the interference factors; (2)Choose scientific and practical methodologies; (3)Standardized operation; (4)Regularly monitor the quality of testing results.

Joint detection of troponin T, high sensitivity C-reactive protein, N-terminal probrain natriuretic peptide applied in the diagnosis of acute coronary syndrome for elderly patients / 中华老年医学杂志

Objective To investigate the value of the joint detection of Troponin T(TnT),highsensitivity C-reactive protein (hs-CRP) and N-terminal probrain natriuretic peptide (NT-proBNP) for the clinical diagnosis of acute coronary syndrome(ACS) in elderly patients.Methods The adequate serum samples were collected in each group:unstable angina (UA) (49 cases),non-ST segment elevations myocardial infarction(NSTEMI) (48 cases),acute myocardial infarction(AMI) (37 cases)and healthy control (45 cases).The levels of TnT and NT-proBNP were measured by electrochemiluminescent double antibody sandwich method,and hs CRP by immune transmission turbidity.The roles of individual and joint detection of the three indicators were analyzed by ROC curve and Logistic regression model.Results Except for TnT in UA group,the serum TnT,NT-proBNP and hs-CRP levels were significantly higher in three ACS groups than in healthy control group (P＜0.05).The largest areas under the ROC curve (AUC) of individual TnT,NT-proBNP,hs-CRP testing and the joint detection for UA were 0.583±0.059,0.786±0.047,0.620±0.058 and 0.787±0.046,for NSTEMI were 0.967±0.022,0.978±0.015,0.897±0.032 and 0.991 ±0.009,for AMI were 0.971 ± 0.024,0.961 ± 0.021,0.874 ± 0.043 and 0.999 ± 0.002,therefore,the area under the ROC curve of the joint detection was increased to some degree as compared with individual TnT,NT-proBNP,hs-CRP testing.Similarly,the best sensitivity and specificity of individual TnT,NT-proBNP,hs-CRP testing and the joint detection for UA were 16.7％ and 100.0％,54.2％ and 91.1％,54.2％ and 75.6％,50％ and 95.6％; for NSTEMI were 93.5％ and 100.0％,95.7％ and 97.8％,67.4％ and 97.8％,95.7％ and 100.0％; for AMI were 94.1％ and 100.0％,91.2％ and 97.8％,67.8％ and 97.8％,100.0％ and 97.8％,respectively.Conclusions Joint detection of TnT,NT-proBNP,hs-CRP can significantly improve the diagnosis of UA; for NSTEMI and other AMI,it can be achieved the optimism of sensitivity and specificity,but its effect of distinguishing NSTEMI and AMI is little.

Determination of serum glucose by isotope dilution liquid chromatography tandem mass spectrometry / 中华检验医学杂志

Objective To develop a candidate reference method for the measurement of serum glucose based on isotope dilution liquid chromatography tandem mass spectrometry(ID-LC/MS/MS)Methods An internal standard [~(13)C_6]glucose was added to serum samples and equilibrated with endogenous glucose.Serum proteins were removed by a precipitation with anhydrous ethanol.Serum glucose and the internal standard were then reacted with 1-phenyl-3-methyl-5-pyrazolone and the formed derivatives were analyzed by liquid chromatography tandem mass spectrometry with multiple reaction monitoring(MRM).The method was calibrated with bracketing calibrators and serum glucose concentrations were calculated by comparing the peak area ratios of samples with that of the calibrators.Results The within-run,between-run and total coefficients of variation averaged 0.36%,0.47%and 0.61%,respectively.The analytical recoveries ranged from 99.0% to 100.9%.Results of analyzing the certified reference material SRM 965a showed an average biases of-0.20%.Conclusions An ID-LC/MS/MS method for measuring serum glucose has been developed.The method is highly precise and accurate and may be used as a candidate reference method.

Determination of serum creatinine by isotope dilution liquid chromatography tandem mass spectrometry: a candidate reference method / 中华检验医学杂志

Objective To develop a candidate reference method for the measurement of creatinine in human serum based on isotope dilution liquid chromatography tandem mass spectrometry (ID-LC/MS/MS). Methods An isotopically labeled internal standard [<'2>H<,3>] creatinine was added to the serum sample and equilibrated with the endogenous creatinine. The samples were treated with anhydrous ethanol to remove proteins by precipitation. After being washed with chloroform for further clean-up, the samples were analyzed by LC/MS/MS. Serum creatinine was quantified by a bracketing calibration. Results The within-run, between-run and total coefficients of variation ranged from 0.52% to 0.61%, 0.11% to 0.59% and 0.61% to 0.83%, and the averages were 0.57%, 0.43% and 0.73%, respectively. The analytical recoveries ranged from 99.09% to 101.13% with an average of 100.3%.The results of analyzing the certified reference material SRM 909b (Level Ⅰ and Ⅱ) and SRM 967b showed biases of less than 0.4%. Conclusions An ID-LC/MS/MS method for measuring serum creatinine has been developed. The method is highly precise and accurate and may be used as a candidate reference method for serum creatinine measurements.

Stability of glycated hemoglobin measurement in whole blood with routine methods / 中华检验医学杂志

Objective To investigate the stability of glycated hemoglobin HbA1c in whole blood sample measured by Tosoh G7, Roche/Hitachi 7170A and NycoCard READER Ⅱ under different storage conditions. Methods Three whole blood samples (EDTA anticoagulated) with different glycated hemoglobin levels and one whole blood sample (heparin anticoagulated) were collected and stored at -80 ℃, -20 ℃, 4 ℃,room temperature(15 -25 ℃), and 37 ℃ HbA1c was analyzed by each method on days 1, 2, 5, 7, 9, 14, 21,28, 35, 42, 49, 56, 63 and 70 respectively. Results The results of sample stored at -80 ℃ appear to be stable for Tesoh G7 and Roche/Hitachi 7170A method. The coefficients of variation (CV) for Tosoh G7 was 0.54%-1.22%. The CV for Roche/Hitachi 7170A was 0.86% -1.82%. When samples was detected with Tosoh G7 method, the results was consistent when the sample was stored at -20 ℃ for 14 days, 4 ℃ for 63 days, room temperature for 5 days, and 37 ℃ for less than 1 day. When samples was detected with Roche/Hitachi 7170A method, the results was consistent when the samples was stored at -20 ℃ for 21 days, 4 ℃ for 42 days, room temperature for 7 days, and 37 ℃ for less than 1 day. The NycoCard READER Ⅱ showed stability at 4 ℃ for 9 days, and room temperature for less than 1 days. Conclusions The stability of whole blood samples is dependent on different methods. Storage time under different temperatures is different.

Determination of serum urea by isotope dilution gas chromatography mass spectrometry:a candidate reference method / 中华检验医学杂志

Objective To develop a candidate reference method for the measurement of urea in human serum based on isotope dilution/gas chromatography/mass spectrometry.Methods [13C,15N2]Urea used as internal standard Was added to the serum sample and equilibrated with endogenous nonlabeled urea.The serum samples were treated with anhydrous ethanol to emove proteins by precipitation.The serum urea and labeled urea were converted into a trimethylsilyl derivative of 2-hydroxypyrimidine and analyzed by gas chromatography/mass spectrometry system with selected ion monitoring.The concentration of serum ureaWas calculated by the theory of bracketing method.Results The average value of within-run oefficient of vailation(CV),between-run CV and total CV of the procedure were 0.38%(ranged from 0.12%to 0.47%),O.62%(ranged form 0.49% to 0.87%)and 0.73%(ranged from 0.51% to 0.93%).Respectively.The analytical recoveries ranged from 99.37% to 100.95%.The resuhs of analyzing the certified refefence material SRM909b(Level Ⅰand Ⅱ)showed a bias less than 0.2%.Conclusion The procedure for measuring urea in serum is a highly accurate and precise method and can be used as a candidate reference method for serum urea assays.

Determination and metrological traceability of glycated hemoglobin / 中华检验医学杂志

This article summarized recent correlative literatures focusing on international standards on glycated hemoglobin.The basic concept,determination of glycated hemoglobin,the present review in laboratory measurement and metrological traceability was introduced.The international community has established reference system and metrological traceability to the International System of Units on HbA1c.Determination in glycated hemoglobin is still in incipient stage in our country.Both clinical laboratorians awareness and clinical determination need to be strengthened.

Evaluation of matrix effects in serum urea measurements / 中华检验医学杂志

Objective To evaluate the matrix effects in serum urea measurements of external quality assessment(EQA)materials and commercial reference materials(calibrators or controls)on enzymatic methods and to verify the trueness of the enzymatic methods.Methods The Clinical and Laboratory Stadards Institute(CLSI)EP 14-A2 protocol was used for the evaluation of matrix effect.An isotope dilution gas chromatography mass spectrometry method was used as the comparative method.Twenty five fresh patient serum samples,15 EQA materials and 13 calibrators or controls were analyzed with 7 enzymatic methods (evaluated methods)and the comparative method and results were processed according to the protocol. The trueness of the evaluated methods were also assessed by comparing the fresh sample results obtained with the evaluated and comparative methods.Results Eight of 15 EQA materials and 3 of 13 calibrators or controls showed no matrix effects on all the 7 routine methods.One processed sample showed matrix effect on all the routine methods.Method dependent matrix effects of other materials were observed on other materials.Calibration biases were also observed on some enzymatic methods.Conclusions Matrix effects and calibration bias have been observed in serum urea measurements.Continued efforts are needed for improving the accuracy and the comparability of serum urea measurements.

Evaluation of matrix effect of processed materials in serum total glycerol measurements / 中华检验医学杂志

Objective To evaluate the matrix effect of processed materials in serum total glycerol measurement and to assess the accuracy of routine test systems.Methods With an isotope dilution liquid chromatography tandem mass spectrometry method as the comparative method，matrix effects of 28 processed materials on 8 routine test systems were evaluated ccording to the NCCLS EP 14 protocol.The processed materials and 20 flesh patient specimens were analyzed with both the comparative method and each of the evaluated methods and results obtained with the two methods were plotted.Two-tailed 95% prediction intervals for the mean of the flesh patient specimen were computed and results on the processed aterials were compared with these intervals for evaluation of matrix effect.Results with the two methods on fresh samples were also compared for assessment of the accuracy of the routine test systems.Results Some of the processed samples showed matrix effects on some of the routine test systems.The observed matrix effects were system-specific and aused either positive or negative biases.Calibration biases were also observed on some test systems.Conclusion Matrix effect and calibration bias have been observed in serum total glycerol measurements.Continued efforts are needed for improving the accuracy of serum total glycerol measurements.