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Objective:To develop a national secondary reference material of Urea and Creatinine in frozen human serum as a standard for metrological traceability.Methods:According to JJF1343-2012 "General and Statistical Principles for Characterization of Reference Materials" and JJF 1006-1994 " Technical Norm of Primary Reference Material ", the homogeneity, stability, and commutability were evaluated;Using the JCTLM recommended methods, the value of the reference materials was assigned through collaboration with 6 accredited reference laboratories from Guangdong Provincial Hospital of Chinese Medicine, Beijing Aerospace General Hospital, Shenzhen Mindray Bio-Medical Electronics, Maccura Biotechnology, Beijing Leadman Biochemistry, and Zhejiang MedicalSystem Biotechnology. Uncertainty components including inhomogeneity, stability and value assignment were evaluated.Results:The results of one-way analysis of variance of homogeneity for the reference materials showed P>0.05, and the stability evaluation was less than the critical value of the t-test. The measured values were in the 95% confidence interval in the four conventional detection systems for commutability, and the certified values and expanded uncertainties were urea:(14.7±0.3) mmol/L ( k=2),Cr:(313.9±14.5) μmol/L ( k=2). Conclusion:The prepared secondary reference materials of urea and creatinine had promising homogeneity, stability, and commutable, the values of urea and creatinine concentration in reference materials were accurate and reliable.
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Objective:To establish a candidate reference method for serum progesterone using isotope dilution liquid chromatography tandem mass spectrometry (ID-LC/MS/MS) in our laboratory, validate the analytic performance of five clinical routine detection systems to explore the comparability of serum progesterone detection by different detection systems.Methods:A candidate reference method for serum progesterone using ID-LC/MS/MS method was established. The sample was pretreated by liquid-liquid extraction method, and the reversed phase liquid phase separation in positive ion mass spectrometry mode was used to detect progesterone in human serum, and the detection time of a single sample was controlled within 5 minutes by gradient elution. In order to improve the accuracy of the method, the bracketing calibration method (BCM) was used to establish the standard curve. The sensitivity, accuracy, precision and specificity of BCM and classical calibration curve method were evaluated according to CLSI C62-A, EP15-A2, EP6-A2 and EP9-A3, and the analytical performance and comparability of five clinical routine progesterone detection systems were evaluated,compared with ID-LC/MS/MS method, the bias at medical decision level 2 and 25 ng/ml was evaluated to see if they were <1/2TEa (12.5%).Results:The limit of detection (LOD) of ID-LC/MS/MS was 0.005 ng/ml. The recoveries of BCM method and classical calibration curve method are 97.95%-101.58% and 96.88%-110.70%, respectively. The measurement results of BCM method for certified reference materials are within its declared uncertainty range. The intra-and inter-assay coefficient of variation ( CV) of BCM method was less than 3.0%, which was better than that of classical calibration curve method ( CV: 2.48%-9.33%). The precision and linear range of the five clinical routine detection systems can meet the detection requirements. The measurement bias of detection system 1, 3 and 5 at 25 ng/ml of medical decision level was less than 1/2TEa, and the measurement bias at 2 ng/ml of medical decision level was more than 1/2TEa. The measurement bias of detection system 2 and 4 at two medical decision levels was less than 1/2TEa. Conclusion:The candidate reference method for serum progesterone ID-LC/MS/MS established in our laboratory meets the requirements of the reference method. BCM has better detection performance than classical calibration curve method. The precision and linearity of the five progesterone clinical detection systems are satisfactory. The five clinical detection systems could meet the clinical requirements at the medical determination level of 25 ng/ml, however, only two of the five clinical detection systems meet the clinical requirements at the medical determination level of 2 ng/ml.
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The concentration and accumulation rate of advanced glycation end products (AGEs) in the body are highly correlated with glycometabolic disorders. Therefore, the clinical detection of AGEs is of great value for the early diagnosis and prognostic evaluation of these diseases. However, due to the complexity of its structure, the diversity of glycosylation sites, and the limitations of existing detection methods, there is still a lack of widely available detection methods in clinical practice. Starting from the structure and classification of AGEs and the value of clinical testing, this article summarizes current status of various laboratory detection methods of AGEs, and the deficiencies and challenges of these testing methods, future directions are further prospected.
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The performance of high sensitivity, specificity and throughput makes liquid chromatography-tandem mass spectrometry (LC-MS/MS) quickly developed and well applicated in clinical laboratories, such as quantitative detection of steroids, vitamins and metabolic small molecules, monitoring drug concertrations as well as the establishing reference methods or verifying reference materials. However, there were still some challenges for the widespread clinical application of LC-MS/MS.
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There are many types of serum tumor markers,and their structures and functions vary.The standardization and harmonization of serum tumor markers will contribute to clinical diagnosis and treatment.Therefore,many scholars are committed to the research of their standardization.However,there are only a few items have been standardized.Due to the complexity of determination,most tumor markers are still facing problems and challenges in the process of achieving standardization.
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Objective To express adenosine deaminase protein by molecular cloning technique.MethodsTotal RNA was extracted from human leukocytes and the cDNA was obtained by reverse transcription.Whereupon the cDNA was used as a template to amplify adenosine deaminase gene by polymerase chain reaction (PCR) and then integrated it into three prokaryotic plasmids pET-28 b, pET-32 a (+) and pHSIE.The plasmid with the correct sequencing was transformed into E.coli BL21 (DE3) by CaCl2 method for the protein expression.The expression activity of these fusion proteins were detected by Western-blot and SDS-PAGE, with the optimized expression conditions.Results Complete fusion of target gene and three prokaryotic plasmids was observed through sequencing.The expressed and accurate ADA protein was identified by Westernblot and SDS-PAGE.The optimal expression conditions were observed:the protein expression would be induced with 0.4 mmol/L IPTG and incubated at 16℃for 24 hours.Conclusion The prokaryotic vectors of adenosine deaminase (BL21+pET-28 b+ADA, BL21+pET-32 a+ADA, BL21+pHSIE+ADA) were successfully constructed and efficiently expressed.
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Renal function test items mainly include creatinine, urea, uric acid, cystatin C, urine-alb, urine protein, α1-microglobulin, β2-macroglobulin and retinol binding protein, etc.The standardization and harmonization of these renal function test items will contribute to clinical diagnosis and treatment.Therefore, many scholars are committed to the standardization of renal function tests and part of these testshave been standardized.But some test items, due to the complexity of the determination, the process of achieving standardization are still facing some problems and challenges.
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Objective To construct the recombinant plasmid pET32a-AKT1 and express human AKT1 protein using prokaryotic expression system .Methods Reverse transcriptase-polymerase chain reaction(RT-PCR) was employed to amplify the gene AKT1 in coding region and integrated it with pET 32a plasmid ,following by transforming it into Escherichia coli DH5α and prokaryotic strains BL21(DE3) .Isopropyl-beta-D-thiogalactopyranoside(IPTG) was adopted to induce its expression .Sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE ) and Western-blot were used for protein identification .Results Complete fusion of target gene and plasmid was observed .The recombinant plasmid pET32a-AKT1 was successfully transferred into the strain DE3 . After IPTG induction ,protein with relative molecular mass 70 000 was expressed by DE3 .Conclusion The recombinant plasmid pET32a-AKT1 is constructed successfully and AKT 1 protein is completely and efficiently expressed by prokaryotic strain DE 3 .
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Objective To discuss the feasibility of enzymatic reference methods in Routine Chemistry external quality assessment (EQA)inlaboratorymedicine.Methods Samplesofthe1stEQAin2012byNationalCenterforClinicalLaboratories(NCCL)and patients′sera were measured by reference methods and 5 clinical analytic systems for the catalytic activity of CK ,LDH ,ALP ,ALT , AST ,GGT and AMY ,then the results of 5 clinical systems were compared with the reference methods′or target value of NCCL by calculating the bias ,and evaluated them according to the criteria of EQA by NCCL .Results The results of EQA samples measured by reference methods was within ± 10% compared with NCCL target value .Compared with the results of reference method ,the through put was 100 .0% for wet clinical chemistry systems measuring both EQA samples and patients′serum ,and the dry clinical chemistry systems was 77 .1 for EQA samples and 97 .1% for patients′serum according to the criterion of EQA ,and the through put was 72 .9% and 63 .6% of wet clinical chemistry systems according to the standard of enzymatic trueness of NCCL .Conclusion Reference method could be applied to EQA ,and will be a great help for the trueness of clinical testing .
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Objective To Evaluate the performance of serum adenosine deaminase assays of different manufacturers and explore the approach for harmonization of test results.Methods It was evaluated the indice including the limit of blank ,precision,linearity range and reference interval of 10 test systems.It was as the reference system by Mindray test system to evaluate the comparability and the difference of ADA results among 10 different systems.The evaluation was performed before and after calibration by a selected fresh serum assigned by the reference system.A commercial calibrator of the minimum matrix effect was selected from 8 different calibrators as the long-term calibrator to harmonize the ADA results of 10 systems.Results The results of LoB were 0.1-6.3 U/L,respectively.The within-run CVs and total CVs of 10 systems were all less than 5%and actual linearity ranges were conformed to claims of manufacturers.After calibration with fresh serum calibrator ,the averaged difference of 10 test systems was reduced from 14% to 3.0%, and the average difference was 1.8% after calibration with long-term calibrator.The common reference interval of all test systems was 5-24 U/L identically.Conclusions The comparability of ADA measurements can be improved by using a common human serum calibrator and the commutable commercial calibrator.And it is necessary and feasible to develop the standardzation of ADA.