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Objective:The allowable total error ( TEa),allowable imprecision ( CV)and allowable bias( Bias)were recommended for 34 routine chemistry analytes in China. Methods:According to the performance specification setting mode newly determined at the Milan conference in Italy,the performance specification was derived based on components biological variation (BV)and current state of the art mode. The data(including EQA data and IQC data)of laboratories participating in the routine chemistry and lipids and lipoproteins EQA activities of the national center for clinical laboratories from 2019 to 2021 was collected through clinet-EQA. For the analytes with biological variation(BV)data,compared the'percentage difference′ of EQA data and the'in-control coefficient of variation of the month′ of IQC data of each research analyte with the three levels evaluation criteria derived based on BV,and calculated the percentage difference passing rate and CV passing rate of all batches in each year. When the passing rate reaches 80%,the performance specifications of this level met the requirements of the recommended performance specifications of the analyte. For the analytes without BV data or analytes whose performance specifications at three levels derived based on BV could not be used as recommended standards,the recommended performance specifications are derived based on the current state of the art. After obtaining the recommended TEa and allowable CV for each analyte,used the formula | Bias|≤ TEa-z? CV to derive the recommended allowable bias. Results:The results of TEa ( CV)% recommended by 34 analytes are as follows:K4.7(2),Na4(1.5),Cl4(1.4),Ca5(2),P9.6(3.9),Glu6.4(2.5),Urea8(3),UA12(4.1),Cre11(3.3),TP5(2),Alb5.2(2.4),TC8.6(2.7),TG13.5(5),HDL-C16.5(4.3),LDL-C20.5(6.2),ApoAⅠ16(5.3),ApoB 17.1(5.5),Lp(a) 24.1(10.4),TBil 12.4(5),DBil 20(7.3),ALT16(5),AST13.5(4.8),ALP17.5(4.8),AMY13.1(3.3),CK11.3(3.8),LDH11(3.9),CHE13.4(5.3),LIP20(6.9),Fe13.3(5.2),Mg14(4.5),Cu17.9(6.8),Zn15.1(6.4),γ-GGT10(3.3),α-HBDH18(5.8).The formula | Bias|≤ TEa-z? CV is used to derive the allowable bias of 34 analytes. Conclusions:For 34 clinical routine chemistry quantitative analytes,the allowable total error,allowable imprecision and allowable bias that meet the current state of the art of Chinese laboratories are recommended.
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Lead poisoning severely threatens human health with its cumulation and durability in the body. The analysis of lead in blood is vital for screening, diagnosis, treatment, and prognostication of lead poisoning and for indirectly monitoring the level of lead in the environment. Although the detection programs are available throughout our country, the accuracy and comparability of the results cannot meet the expectation. A variety of factors can affect the accuracy of blood lead testing. To promote the application of blood lead analysis in clinical trials and reduce the bias of results, a better reference system for blood lead analysis should be established to evaluate the accuracy of traditional methods, promote the standardization of blood lead analysis and achieve accurate blood lead testing.
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Objectives:To establish a candidate reference measurement procedure based on isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) for cyclosporin A, tacrolimus, sirolimus, and everolimus measurements in human whole blood.Methods:The isotope labeled cyclosporine A, tacrolimus, sirolimus, and everolimus were selected as the internal standards. Samples were accurately weighed while protein precipitation and solid phase extraction were selected for the sample preparation. The standard curve method was applied for quantification. The ultra-high liquid chromatography coupled with triple quadrupole mass spectrometer was used for analysis. The specificity, matrix effect, detection limit, quantification limit, precision, accuracy, and uncertainty of the method were evaluated.Results:The method showed good selectivity and specificity. No apparent interferences or matrix effects were found in the target analyte measurements. The detection limits and quantification limits of cyclosporin A, tacrolimus, sirolimus and everolimus met clinical requirements. Intra-batch coefficients of variation ( CV) were from 1.4% to 1.8% for CSA, from 1.7% to 2.8% for TAC, from 1.3% to 3.7% for SRL and from 2.3% to 3.2% for EVR, and total CVs were from 1.8% to 2.9% for CSA, from 1.7% to 3.8% for TAC, from 2.6% to 4.7% for SRL and from 3.5% to 4.6% for EVR. The relative recoveries were from 97.9% to 100.3% for CSA, from 98.4% to 103.1% for TAC, from 99.4% to 102.0% for SRL and from 98.3% to 99.4% for EVR, and the relative expanded uncertainties at four concentrations were from 4.2% to 4.4% for CSA, from 1.5% to 2.4% for TAC, from 4.4% to 4.9% for SRL and from 2.2% to 2.7% for EVR. Conclusion:A candidate reference measurement procedure for the cyclosporine A, tacrolimus, sirolimus, and everolimus in human whole blood was established by ID-LC-MS/MS.
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Objective:To construct a prokaryotic expression vector for human retinol binding protein 4 (hRBP4) that allows technicians to obtain hRBP4 purified protein with low cost, high efficiency, high concentration and high purity.Methods:The hRBP4 coding sequence provided by National Center for Biotechnology Information was optimized by E. coli codons, and a synthetic DNA fragment was cloned into the PET-28A (+) prokaryotic expression vector to construct a recombinant hRBP4 expression plasmid. The recombinant protein was transformed into E. coli BL21, and the induced expression conditions (temperature, rotate speed and isopropyl β-d-thiogalactoside concentration) were optimized. The recombinant protein was purified by His fusion tag. Results:The recombinant hRBP4 prokaryotic expression plasmid was successfully constructed, and the expression concentration and induction temperature of the recombinant protein were optimized. The results of sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that a band with a relative molecular weight of 26 000 daltons was clearly visible in the purified product. The purified hRBP4 protein could be detected clinically, and there was a good linear relationship between the dilution ratio and the detection concentration.Conclusions:The recombinant hRBP4 protein has high purity, high concentration, and short production cycle. It has the potential to become a candidate for reference materials for laboratory quality evaluations.
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Objective:To investigate the molecular mechanism of proliferation-inhibiting effect of icaritin on hepatoma cells via regulating miRNA-329 (miR-329) and miRNA-1236 (miR-1236).Methods:Hepatoma cell line HepG2 was treated with icaritin at different concentrations (2.5, 5.0, 10.0, 20.0, 40.0 μmol/L), and the control group only added dimethyl sulfoxide (DMSO). The half inhibitory concentration ( IC50) of icaritin on HepG2 cells and cell proliferation rate were detected by CCK-8 after cultured for 36 h. HepG2 cells were treated with 400 μg/L alpha fetoprotein (AFP). After cultured for 0, 12, 24, 36, 48 and 60 h, the effect of AFP on the proliferation of HepG2 cells was detected by CCK-8 method. AFP-3'UTR reporter plasmid pmirGLO-AFP-3'UTR plasmid was constructed, pmirGLO blank vector plasmid, pmirGLO-AFP-3'UTR plasmid, miR-329 or miR-1236 mimics or inhibitors, control plasmid of mimics (NC), control plasmid of inhibitors (INC) were respectively co-transfected with HepG2 cells, and the effect of miR-329 and miR-1236 on the luciferase activity was detected by dual-luciferase reporter assay after cultured for 24 h. Western blot and real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) were used to detect the effects of icaritin on the expressions of AFP, miR-329 and miR-1236 in HepG2 cells. HepG2 cells were respectively transfected with mimics and inhibitors of miR-329 and miR-1236 to detect the effects of miR-329 and miR-1236 on the expression of AFP. Results:The cell proliferation rates of 2.5, 5.0, 10.0, 20.0, 40.0 μmol/L icaritin group and control group were (80.4±2.3)%, (73.2±1.6)%, (51.7±3.3)%, (38.2±4.6)%, (29.5±4.3)%, and (94.0±2.9)%, respectively, and the difference was statistically significant ( F = 75.65, P < 0.01); the differences in the proliferation rate of HepG2 cells between different concentrations of icaritin group and control group were statistically significant (all P < 0.01). The IC50 of icaritin on HepG2 cells was 10 μmol/L. The relative expressions of AFP mRNA in 2.5, 5.0, 10.0, 20.0, 40.0 μmol/L icaritin group and control group were 0.83±0.06, 0.69±0.02, 0.53±0.07, 0.45±0.01, 0.33±0.07, and 1.00±0.01, respectively, and the difference was statistically significant ( F = 42.67, P < 0.01); the differences in the relative expressions of AFP mRNA between different concentrations of icaritin group and control group were statistically significant (all P < 0.01). HepG2 cells were treated by 400 μg /L AFP for 0, 12, 24, 36, 48 and 60 h, and the cell proliferation rates were (102±5)%, (138±13)%, (186±24)%, (260±12)%, (311±15)%, and (348±25)%, respectively, and the difference was statistically significant ( F = 27.483, P < 0.01); the differences in the cell proliferation rate between different time of AFP treatment and 0 h were statistically significant (all P < 0.01). Compared with the control group, different concentrations of icaritin can promote the expression of miR-329 and miR-1236 (all P < 0.01). After co-transfection of miR-329 and miR-1236 mimics and AFP-3'UTR, the luciferase activity decreased by about 40%; after co-transfection of miR-329 and miR-1236 inhibitors and AFP-3'UTR, the luciferase activity increased about 1.5 times. Both miR-329 and miR-1236 can reduce the expression levels of AFP protein and mRNA (both P < 0.05). Conclusion:Icaritin can promote the binding of miR-219, miR-1236 and AFP-3'UTR by promoting the expression of miR-329 and miR-1236, inhibit the stability and translation activity of AFP mRNA, inhibit the expression of AFP, and thus inhibit the proliferation of hepatoma cells in vitro.
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Serum procalcitonin (PCT) is an infectious biomarker that is widely used in clinical practice. Point-of-care testing (POCT) as a detection technology has been increasingly used in clinical application. This paper discussed the current status and problems in the application of POCT in PCT, with a view to improving the quality of diagnostic products in POCT.
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Objectives:To establish a candidate reference method of isotope dilution liquid chromatography tandem mass spectrometry (ID-LC/MS/MS) for the determination of human plasma normetanephrine, and to evaluate the performance of the method. The method was used to quantify the samples of the external quality assessment program, and to initially evaluate the detection status of plasma normetanephrine.Methods:The isotope standard solution of normetanephrine was selected as the internal standard, the gravimetric method was used for sampling, and the standard curve method was used for quantification. Protein precipitation combined with weak cation solid phase extraction was used for pretreatment, and ultra-high liquid chromatography-coupled triple quadrupole mass spectrometry was used for LC/MS analysis. According to the relevant EP documents, the specificity, matrix effect, detection limit, quantification limit, precision, accuracy, and uncertainty of the method were estimated. This method is used to quantify the samples of the 2020 National Center for Clinical Laboratories external quality assessment program of normetanephrine. Taking the average value of this method as the target value, the optimal allowable total error standard of biological variation as the evaluation limit, the quality of the laboratory testing was evaluated.Results:The method had good specificity, and the interferences and matrix effects did not affect the detection results. The detection limit and quantification limit of plasma normetanephrine were 1.08 pg/g and 3.54 pg/g, respectively. The intra-batch coefficient of variation ( CV) and total CV were 0.43%-1.10% and 0.61%-1.42%, respectively. The relative recovery rates were 98.5%～101.9%. The relative expansion uncertainty of the four plasma samples were 3.10%, 2.34%, 2.16%, and 1.73%, respectively. The results of the external quality assessment program showed that the pass rates of the 202013 and 202014 samples were 80% and 85%, respectively. Conclusions:The study established a candidate reference method of ID-LC/MS/MS for the measurement of plasma normetanephrine. The method is accurate, precise and simple, and is expected to be used as a reference method for the determination of plasma normetanephrine, and can be applied to quantify the samples of the external quality assessment program.
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Objective:To establish the allowable total error (TEa) of the national external quality assessment (EQA) program in line with the current quality level of serum folate measurement in China.Methods:The data of serum total folate test in the clinical laboratory of a hospital in Beijing in 2016 were collected, and the Stata SE 15 software was used for Monte Carlo simulation to obtain the false-negative rate under different bias and inaccuracy conditions. The Origin Pro 9.1 software was used to make the contour figure. The TEa of serum total folate test is derived based on the acceptable false-negative rate. National EQA data of serum total folate in 2020 were collected to calculate the pass rate of participating laboratories and the laboratory pass rate of quality control products at each level under the five TEa derived from the analysis performance on clinical outcomes, biological variation, and the evaluation criterion of national EQA.Results:Based on the influence of analytical performance on clinical outcomes, the TEa was 10%. Under this TEa, the pass rate of the first EQA program of serum total folate in 2020 was more than 80%, and the pass rate of the second time was 73.1%. Under the minimum (46.57%) and appropriate level of TEa (15.52%) derived from biological variation and national EQA evaluation criterion, the pass rate of serum total folate in the two EQA programs in 2020 exceeded 85%.Conclusion:The analytical performance of serum total folate in China cannot meet the requirements of TEa derived based on the effect of analytical performance on clinical outcomes. An appropriate level of TEa derived based on biological variation (15.52%) is suggested as the recommended criterion for the TEa of serum total folate test.
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Accurate determination of drug concentration in blood samples is a necessary prerequisite for therapeutic drug monitoring (TDM) and the implementation of precise drug treatment, and it is also one of the important tasks of clinical laboratories.TDM plays an important role in clinical treatment in immunosuppressants (cyclosporine A, tacrolimus), psychotropic drugs (valproic acid, carbamazepine) and other drugs that require monitoring of drug concentration. There are many types of methods used for TDM for the detection of drug concentration in blood samples. At present, only a few immuno-assay methods were approved for marketing with detection systems and kits, most methods used for TDM are high performance liquid chromatography or liquid chromatography-tandem mass spectrometry which belong to laboratory developed tests (LDTs). Detection of TDM samples has many problems, such as incomparable testing results and large bias between different testing systems, including the biasbetween both different methods and different laboratories using the same method. There are several reasons:(1) the traceability chain has not been established, (2) the methods have not yet been standardized, (3) the coverage of the EQA plan is insufficient, (4) the awareness of TDM laboratories to participate in the EQA plan is insufficient, (5) TDM standardization is still in its infancy. These problems restrict the clinical application of TDM and the development of related research work. In order to solve these problems, it is necessary to: (1) Establish a reference system to realize the traceability of the test results; (2) While gradually increasing the TDM EQA plan items, the Trueness evaluation plan should be carried out as soon as possible; (3) Standardized TDM sample testing Technology; (4) Strengthen laboratory management and establish a complete quality management system.
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Background@#Using commutable external quality assessment (EQA) materials is important for monitoring successful harmonization efforts. We assessed the commutability of four human serum pool (HSP) preparations to identify candidate EQA materials for alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity measurement. @*Methods@#One set each of 85 clinical samples (CSs) was collected for ALT and AST activity measurement. The 15 candidate EQA materials included four types of HSP preparations (A to D): materials A, C, and D contained human original recombinant (HOR) aminotransferases; materials B was mixed leftover samples. The CSs and 15 candidate EQA materials were analyzed using seven routine assays, and the ln-transformed results were analyzed in 21 assay pairs. Commutability was assessed using Deming regression, with a 95% prediction interval (CLSI approach) and the difference in bias with an error component model (International Federation of Clinical Chemistry and Laboratory Medicine [IFCC] approach). @*Results@#For ALT, all materials were commutable for 14–21 assay pairs according to the CLSI and IFCC approaches. For AST, B01-03 showed commutability for 14-21 assay pairs, and C01-03 and D01-03 showed commutability for no less than 10 assay pairs according to the two approaches. A01-06 were commutable for 9-16 assay pairs according to the CLSI approach, but for 6-9 assay pairs according to the IFCC approach. @*Conclusions@#Mixed leftover samples showed desirable commutability characteristics as candidate EQA materials for routine aminotransferase activity measurements. Human serum bases supplemented with HOR were commutable for most routine ALT activity measurements.
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Objective@#The aim of this study is to evaluate the commutability of 16 processed materials for 17-hydroxyprogesterone by using 2 commutability assessment approaches.@*Methods@#52 serum specimens were collected in Clinical Laboratory Department of Beijing Hospital from February 2018 to June 2019. According to the report of the Clinical and Laboratory Standards Institute (EP14-A3) document and the recommendations of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) working group on commutabilityassessment, serum 17-hydroxyprogesterone isotope diluent chromatogram tandem mass spectrometry (ID-LC/MS/MS) was used for comparison. Three clinical routine analysis systems (1 radioimmunoassay, 2 LC/MS analysis methods) were used to determine the concentration of 17-hydroxyprogesterone in 52 human serum samples and 16 processed materialsfor commutabilityassessment.@*Results@#Combined with the results of the two commutability assessment, all accuracy verification materials and national steroid hormone standards showed good commutability in the LC/MS analysis system, and 6/9 EQA materials showed commutability in the three routine analysis systems.All materials showed good commutability in the LC/MS analysis system of bias difference method.@*Conclusions@#The two kinds of commutability assessment results are different. Bias difference method has more clinical value, but it has certain application limitations. The use of fresh frozen human serum as a quality assessment materialfor serum 17-hydroxyprogesterone is meets the commutability requirement.
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Objective:To establish an accurate quantitative method for the determination of serum glycated albumin (GA) based on isotope dilution liquid chromatography tandem mass spectrometry (ID-LC/MS/MS).Methods:This study optimized the ID-LC/MS method recommended by the Japan Society of Clinical Chemistry (JSCC). Albumin (Alb) was purified by anion exchange chromatography. The standard solution of purified sample, lysine (Lys) and deoxyfructosyl lysine (DOF-Lys) and their mixed internal standard were weighed accurately by weight method. The Alb was hydrolyzed to amino acid, and Lys and DOF-Lys were detected to determine GA. The method was evaluated and compared with the routine method.Results:The purity of Alb was 100%. The R 2 ranges of Lys and DOF-Lys linear standard curves were 0.997 8-0.999 9 and 0.999 4-1.000 0. The results of three-day precision evaluation of low, medium and high concentrations of serum were 0.69%-1.97%, 1.13%-2.33% and 1.37%-2.54% in intra, inter and total Coefficient of variation ( CV). In the accuracy evaluation, the deviations between the results of three concentration standards and the certified value were -2.56%, -1.87% and-2.94%. The matrix effects of Lys and DOF-Lys were 89.92% and 109.98%, and the carryover rates were -1.13% and -3.27% respectively. The detection limit and quantitative limit of Lys were 0.075 nmol/g and 0.248 nmol/g, and the DOF-Lys were 0.007 nmol/g and 0.024 nmol/g. The relative expanded uncertainty was 1.60%-2.03%. The fitting regression curve R 2 with the routine method was 0.970 7. Conclusions:A method was established for the detection of serum GA. The method evaluation was accurate and reliable. It was expected to be a candidate reference method for the detection of serum GA.
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Objective:To establish a candidate reference method for simultaneous determination of whole blood iron and copper based on ICP-MS technology.Methods:Cobalt (Co) was chosen as the internal standard, and was added gravimetrically into the standard solution and the whole blood sample. The whole blood sample was digested by electric heating plate and diluted. The isotopic ratios of 57Fe/ 59Co and 63Cu/ 59Co were measured by ICP-MS in the isotopic analysis mode. The precision, standard recovery and accuracy of the method were verified by measuring EQA materials and certified standard materials, and the performance of the method was further evaluated by method comparison. Results:The detection limit of iron is 0.136 mg/kg and the limit of quantification was 0.454 mg/kg in this method. The detection limit of copper in whole blood was 0.008 mg/kg and the limit of quantification was 0.027 mg/kg. The standard curve of copper ranges from 0.83-3.33 mg/kg, iron ranges from 167-667 mg/kg. The calibration curve of iron and copper was good linearity ( R 2>0.999 90). The precision of the method did well. The total CV range was 0.42%-0.68% for iron and 0.14%-0.94% for copper. The spike recoveries were all around 100%, the range of iron: 99.69%-100.07%; copper: 99.26%-100.51%. The correlation between this method and the existing clinical mass spectrometry methods was good. Conclusions:A candidate reference ICP-MS method for simultaneous determination of whole blood Fe and Cu was established. This is a simple and rapid method with good precision and spike recovery compared to the traditional one.
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Objective:To evaluate the performance of aldosterone testing in China through the External Quality Assessment (EQA) and improve the testing quality of aldosterone.Methods:Two kinds of EQA program for aldosterone were carried out in China, one of which is Routine EQA and the other is Trueness verification scheme. Lyophilized sera with 5 concentration levels were used as quality control of Routine EQA. The results were grouped according to the instrument. Target values and the coefficient of variation ( CV) were calculated in each group. Trueness verification scheme was verified by using frozen human sera of 3 concentration levels determined by the reference method, and the bias of each instrument group from the target value was calculated. Results:272 laboratories submitted the testing results, and 91.6% of laboratories used chemiluminescence method. The maximum CV was obtained by radioimmunoassay and liquid chromatography mass spectrometry, and the robust CVs were 14.6%-33.4% and 43.5%-53.9%, respectively. For chemiluminescence methods, the robust group CV was less than 10%. The results of the Trueness verification scheme showed that liquid chromatography mass spectrometry method was the most accurate method, with biases of -7.9%, 8.9% and -0.7% for the three quality controls. Diasorin system had the more accurate results deviated from the target by 58.7%, 7.9% and -2.1%, respectively. The results of other chemiluminescence methods were negatively correlated with the sample concentration, and one of them with a bias of 479%. Conclusions:The accuracy and comparability of aldosterone among laboratories in China are not satisfactory. Reagent manufacturers and laboratories should pay more attention to EQA, with the aldosterone results traceable to SI unit, and improve the test quality of aldosterone.
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Catecholamines include dopamine, norepinephrine and epinephrine. The main metabolites are 3-methoxytyramine, normetanephrine, metanephrine,homovanillic acid and vanillylmandelic acid. Detection of catecholamines and their metabolites is the cornerstone for the diagnosis of neuroendocrine tumors derived from neural crest such as pheochromocytoma, paraganglioma and neuroblastoma. Liquid chromatography tandem mass spectrometry has been widely used in the detection of catecholamines and their metabolites due to its high sensitivity and high specificity. However, the results of liquid chromatography tandem mass spectrometry in different laboratories are quite different and lack comparability. Accurate determination of catecholamines and their mtabolites in plasma and urine is currently a challenge in the field of clinical detectionbecause of their susceptibility to oxidative degradation, presence of numerous interferences and low concentration in plasma and urine samples.
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Aldosterone and renin are important clinical indicators in the diagnosis and treatment of hypertension. There are two kinds of methods for aldosterone detection, including immunoassay and mass spectrometry. The coefficients of variation within the methods, as well as the deviation between methods are large.And the results of immunoassay are much higher than that of mass spectrometry. Clinically, renin tests mainly include renin concentration and renin activity, both of which have advantages and disadvantages in the diagnosis and treatment of hypertension. The disordered test results of aldosterone and renin brings great trouble to the clinical diagnosis and treatment. Standardization/consistency is urgently needed to make the test results of each laboratory more accurate and comparable. The standardization project for renin and aldosterone in China will greatly promote the standardization process in order to provide accurate and comparable clinical tests.
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Objective:The aim of this study is to evaluate the commutability of 16 processed materials for 17-hydroxyprogesterone by using 2 commutability assessment approaches.Methods:52 serum specimens were collected in Clinical Laboratory Department of Beijing Hospital from February 2018 to June 2019. According to the report of the Clinical and Laboratory Standards Institute (EP14-A3) document and the recommendations of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) working group on commutabilityassessment, serum 17-hydroxyprogesterone isotope diluent chromatogram tandem mass spectrometry (ID-LC/MS/MS) was used for comparison. Three clinical routine analysis systems (1 radioimmunoassay, 2 LC/MS analysis methods) were used to determine the concentration of 17-hydroxyprogesterone in 52 human serum samples and 16 processed materialsfor commutabilityassessment.Results:Combined with the results of the two commutability assessment, all accuracy verification materials and national steroid hormone standards showed good commutability in the LC/MS analysis system, and 6/9 EQA materials showed commutability in the three routine analysis systems.All materials showed good commutability in the LC/MS analysis system of bias difference method.Conclusions:The two kinds of commutability assessment results are different. Bias difference method has more clinical value, but it has certain application limitations. The use of fresh frozen human serum as a quality assessment materialfor serum 17-hydroxyprogesterone is meets the commutability requirement.
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Objective:To investigate the molecular mechanism of alpha-fetoprotein (AFP) inhibiting cisplatin-induced apoptosis of hepatocellular carcinoma cells.Methods:HepG2 (AFP positive) and QSG-7701 (AFP negative), two common hepatocyte carcinoma cell lines were selected. The expression of AFP was knockdown in HepG2 cells with RNA interference method and AFP expression plasmid was transfected in QSG-7701 cells. After the cells were cultured for 12, 24, 36 and 48 hours, the cell proliferation was detected by cell counting kit-8 (CCK-8) assay. After HepG2 and QSG-7701 cell lines were interfered or overexpressed AFP protein for 24 h, respectively, apoptotic inducer cisplatin (CDDP) was added. The effect of AFP on apoptosis induced by CDDP in hepatocellular carcinoma cells was determined by flow cytometry. The interaction between AFP and transcription factor retinoic acid receptor (RAR) was examined by protein coimmun oprecipitation (CoIP) technique. The effect of AFP expression level on the expression of DNA damage inducible transcript 1 ( DDIT1), and the effects of AFP and DDIT1 on apoptosis of hepatocellular carcinoma cells were tested by chromatin immunoprecipitation (ChIP) assay and Western blotting. T test was performed for statistical analysis. Results:The results of CCK-8 test showed that after plasmid transfected for 12, 24, 36 and 48 hours, the relative proliferation rates of QSG-7701 cells overexpressed AFP increased by 28.7%±2.7%, 49.8%±6.1%, 65.8%±3.0% and 79.3%±2.0%, respectively; however the relative proliferation rates of HepG2 cells after AFP knockdown decreased by 16.5%±6.1%, 28.5%±5.7%, 42.5%±1.7% and 57.6%±3.8%, respectively, when compared with the control group, and the differences were statistically significant ( t=3.978, 4.357, 3.461, 3.636, 2.858, 2.446, 3.233 and 4.492, all P<0.05). The results of flow cytometry indicated that after AFP overexpression for 24 and 48 hours, the apoptosis rate of QSG-7701 cells decreased by 46.3%±2.9% and 47.7%±7.4%, respectively, compared with cisplatin induced cells; however after AFP knockdown for 24 and 48 hours, the apoptosis rate of HepG2 cells increased by 86.7%±4.0% and 31.6%±10.5%, respectively, compared with cisplatin induced cells, and the differences were statistically significant ( t=3.543, 3.893, 2.336 and 2.561, all P<0.05). The results of CoIP experiment showed that AFP could interact with RAR. After AFP knockout in HepG2 cells, after nucleoprotein extracted, RAR entering the nucleus increased as compared with the control group. However, after overexpression of AFP in QSG-7701 cells, RAR entering the nucleus decreased compared with the control group. The results of ChIP experiments showed that AFP could regulate the expression of DDIT1. The expression of DDIT1 in AFP knockdown HepG2 cells was higher than that of control group, however the expression of DDIT1 in AFP overexpressed HepG2 cells was lower than that of control group. Compared with the control group, the apoptosis rate of HepG2 and QSG-7701 cells increased by 53.1%±4.0% and 73.3%±6.4% respectively after transfecting with DDIT1, and the differences were statistically significant ( t=3.462, 3.012, P=0.009, 0.017). In QSG-7701 cells, after AFP overexpression, the apoptosis rates decreased by 46.6%±4.8% compared with cisplatin added alone. After overexpression of AFP, cisplatin added and overexpression of DDIT1, the apoptosis rate increased by 43.6%±2.7% as compared with that of overexpression of AFP and cisplatin added, and the differences were statistically significant ( t=2.833 and 2.545, P=0.018 and 0.029). In HepG2 cells, after AFP knockdown the apoptosis rate increased by 73.3%±6.1% compared with cisplatin added alone; and after AFP knockdown, cisplatin added and DDIT1 knockdown the apoptosis rate decreased by 32.7%±3.7% as compared with AFP knockdown and cisplatin added, and the differences were statistically significant ( t=2.497 and 2.773, P=0.032 and 0.020). Conclusions:AFP can inhibit the expression of its downstream gene DDIT1 by interacting with the transcription factor RAR, which not only promotes the proliferation of hepatocellular carcinoma cells but also enhances the anti-apoptosis ability of hepatocellular carcinoma cells and weakens cisplatin induced apoptosis in hepatocellular carcinoma cells.
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BACKGROUND: Accurate serum total thyroxine (TT4) measurement is important for thyroid disorder diagnosis and management. We compared the performance of six automated immunoassays with that of isotope-diluted liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) as the reference method. We also evaluated the correlation of thyroid stimulating hormone (TSH) with TT4 measured by ID-LC-MS/MS and immunoassays. METHODS: Serum was collected from 156 patients between October 2015 and January 2016. TT4 was measured by immunoassays from Abbott (Architect), Siemens (ADVIA Centaur XP), Roche (E601), Beckman-Coulter (Dxi800), Autobio (Autolumo A2000), and Mindray (CL-1000i), and by ID-LC-MS/MS. Results were analyzed using Passing-Bablok regression and Bland-Altman plots. Minimum requirements based on biological variation were as follows: a mean bias of ≤4.5% and total imprecision (CV) of ≤3.7%. RESULTS: All immunoassays showed a correlation >0.945 with ID-LC-MS/MS; however, the slope of the Passing-Bablok regression line varied from 0.886 (Mindray) to 1.23 (Siemens) and the intercept from −12.8 (Siemens) to 4.61 (Mindray). Only Autobio, Beckman-Coulter, and Roche included the value of one in the 95% confidence interval for slope. The mean bias ranged from −10.8% (Abbott) to 9.0% (Siemens), with the lowest value noted for Roche (3.5%) and the highest for Abbott (−10.8%). Only Abbott and Roche showed within-run and total CV ≤3.7%. CONCLUSIONS: Though all immunoassays correlated strongly with ID-LC-MS/MS, most did not meet the minimum clinical requirement. Laboratories and immunoassay manufacturers must be aware of these limitations.
Subject(s)
Humans , Bias , Diagnosis , Immunoassay , Mass Spectrometry , Methods , Thyroid Gland , Thyrotropin , ThyroxineABSTRACT
Objective Accurate measurement of aldosterone is critical in the diagnosis of primary aldosteronism. We compared the harmonization of three assays including isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) and two chemiluminescence immunoassays (CLIAs:system A and system B) for the aldosterone measurement. Methods A total of 45 plasma samples, 4 quality control materials, 5 lyophilized bovine serums, and 3 fresh frozen human serum pools were measured by three assays respectively. Based on CLSI EP15-A3 rule, the precision was assessed by coefficient of variance. Deming regression and Bland&Altman plots was performed for method comparison, and correlation coefficient was calculated for concordance (CCC). Results All three methods met the performance criteria based on desirable biological variation for precision (<7.35％). System A showed a relevantly good correlation and comparability with ID-LC/MS/MS (R2=0.985, CCC=0.967), while System B showed relevantly bad correlations and comparability with both System A (R2=0.538, CCC=0.605) and ID-LC/MS/MS (R2=0.547, CCC=0.528).. However, the average relevant bias of two CLIAs exceeded the bias requirement derived from biological variation (18.60％). Conclusion Significant differences were found in the measurement of plasma aldosterone using ID-LC-MS/MS and two CLIAs, which urges the establishment of traceability hierarchy and improvement of reagents' specificity for standardization of aldosterone measurement in clinical settings.