Development of human serum matrix-based unconjugated estriol-certified reference material by recommended ID-LC-MS/MS reference method.

To solve long-term lack of traceability of commercial calibrator kits and standardize clinical routine assays, we developed a human serum matrix-based unconjugated estriol (uE3) reference material (RM) with five concentration gradients. The RMs of uE3 were certified by the National Institute of Metrology (NIM) with the codes of GBW (E) 091048, GBW (E) 091049, GBW (E) 091050, GBW (E) 091051, and GBW (E) 091052. The RMs were determined by isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) reference method which was developed in our group and recommended by the Joint Committee on Traceability on Laboratory Medicine (JCTLM). GBW09224 is intended for use as a primary reference material to enable the SI-traceable measurement of uE3. This study describes the development process of these certified RMs. The candidate material was prepared by collecting from the remaining serum samples after routine clinical testing. Satisfactory homogeneity and stability were shown in these RMs. They are also commutable between the reference method and the three routine clinical immunoassay systems. To improve the accuracy of value assignment, a collaborative study in nine reference laboratories was conducted which was performed according to ISO/WD 15725-1 and all of the reference laboratories have been confirmed by China National Accreditation Service for Conformity Assessment (CNAS). The raw results were statistically analyzed and processed, coupled with uncertainty evaluation, to obtain the certified value: GBW (E) 091048 is 22.1 ± 1.3 nmol/L, GBW (E) 091049 is 33.6 ± 1.6 nmol/L, GBW (E) 091050 is 10.4 ± 0.8 nmol/L, GBW (E) 091051 is 15.5 ± 1.0 nmol/L, GBW (E) 091052 is 47.0 ± 2.0 nmol/L. The preparation process of human serum matrix-based reference material and the lack of these type of secondary (commutable) reference material of unconjugated estriol lead to the interruption of its traceability chain, which is a problem to be solved in its standardization as mentioned in the metrological traceability in ISO 17511, 2020.

Expression, purification and identification of isotope-labeled recombinant cystatin C protein in Escheichia coli intended for absolute quantification using isotope dilution mass spectrometry.

Isotope-labeled proteins are expected to be used as internal standard proteins in the field of protein quantification by isotope dilution mass spectrometry (ID/MS). To achieve the absolute quantification of Cystatin C (Cys C) based on ID/MS, we aims to obtain 15N isotope-labeled recombinant Cys C (15N-Cys C) protein. Firstly, the Cys C gene was optimized based on the preferred codons of Escherichia coli, and inserted into the pET-28a(+) expression plasmid. Then, the plasmid was transformed into TOP10 and BL21 strains, and 15N-Cys C was expressed in M9 medium using 15N as the only nitrogen source. 15N-Cys C was detected by SDS-PAGE, protein immunoblotting and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). The characteristic peptides obtained from 15N-Cys C were analyzed by a Q Exactive Plus MS system. Results showed that 53.06% of the codons were optimized. The codon adaptation index of the Cys C genes increased from 0.31 to 0.95, and the GC content was adjusted from 64.85% to 54.88%. The purity of 15N-Cys C was higher than 95%. MALDI-TOF MS analysis showed that the m/z of 15N-Cys C had changed from 13 449 to 14 850. The characteristic peptides showed that 619.79 m/z (M+2H)2+ was the parent ion of 15N-Cys C and that the secondary ions of 15N-labeled peptides from y+5 to y+9 were 616.27 m/z, 716.33 m/z, 788.39 m/z, 936.43 m/z, and 1052.46 m/z, respectively. In conclusion, we successfully expressed, purified and identified of 15N-Cys C protein in Escheichia coli intended for absolute quantification using ID/MS.

An isotope dilution liquid chromatography-tandem mass spectrometry candidate reference measurement procedure for aldosterone measurement in human plasma.

Accurate quantitation of aldosterone is clinically important in standardized testing for primary aldosteronism. The results are often variable when performed by clinical immunoassays. To standardize and ensure the accuracy of clinical systems, reference measurement procedures (RMPs) with higher metrological order are required. A simple and reliable isotope dilution LC-IDMS/MS-based measurement procedure for human plasma aldosterone has been developed. This method involved plasma spiked with a deuterium-labelled internal standard, equilibrated for 0.5 h, and extracted by liquid-liquid extraction (LLE) without derivatization. Aldosterone and its structural analogues were baseline separated with a C18-packed UHPLC column with gradient elution within 7 min. The signal intensity variability and measurement imprecision were reduced by bracketing calibration during plasma aldosterone value assignment. The limit of detection (LoD) was 19.4 pmol/L with a signal-to-noise ratio (S/N) > 3. The lowest limit of quantification (LLoQ) was 27.7 pmol/L (S/N > 10 and CV < 10.0%). LLE was performed with 1 mL of n-hexane/ethyl acetate (3:2, v/v), and the extraction recovery was determined to be 92.15 ± 3.54%. The imprecisions were ≤ 3.18% for samples at 124.8, 867.0, and 2628.5 pmol/L. The recoveries were 98.11-101.61%. The relative bias between this candidate RMP and the established RMP was 2.76-1.89%. The linearity response ranged from 27.7 to 2774.4 pmol/L with R2 = 0.999. The method performance met the requirements of RMPs (≤ 5% total CV and ≤ 3% bias). Furthermore, the developed method was applied to evaluate immunoassays through 41 patient sample comparisons. The calibration and measurement capability (CMC) of this method were also evaluated by measuring these samples. The candidate RMP can serve as an accurate reference baseline for routine methods and can be used for value assignment for reference materials. Selected ion chromatograms by LC-MS/MS using a C18 column for aldosterone and its structural analogues.

Performances Evaluation of Four Systems for Homocysteine Determination by LC-MS/MS Reference Method.

BACKGROUND: The aim of this study is to verify the analytical performance of four homocysteine detection systems made in China and to explore the comparability of homocysteine detection systems by isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) reference method. METHODS: The intra-batch precision, inter-batch precision, accuracy, and linear range of four homocysteine detection systems were evaluated. The ID-LC-MS/MS reference method was used to evaluate the comparability and accuracy of fresh frozen serum samples in four different detection systems of homocysteine. The ID-LC-MS/MS reference method is used to assign samples as calibrators to calibrate each system. The variation and deviation of fresh serum samples between different systems before and after calibration were compared. RESULTS: The intra-batch imprecision of the four detection systems was less than 5%, and the coefficient of variation of inter-batch imprecision was less than 6.7%. The precision met the clinical requirements. Before calibration, the results measured by detection system 2 are consistent with the ID-LC-MS/MS reference method, which meets the requirements of accuracy verification. The regression equation of R² ≥ 0.975 in the regression equation of linear analysis of the four systems, the linearity of the four detection systems is good in the range of evaluation concentration, and all of them can meet the declared linear range. The absolute average bias of fresh serum measured by the four detection systems after calibration decreased from 3.76 µmol/L, 0.96 µmol/L, 1.30 µmol/L, -1.56 µmol/L to 0.31 µmol/L, 0.28 µmol/L, 0.4 µmol/L, 0.40 µmol/L, respectively. The relative average bias decreased from 22.6%, 7.50%, 11.0% and -8.50% to 1.98%, 1.78%, 2.59%, 2.34%, respectively. After calibration, the slope and intercept of the regression curve of the fresh serum measured by the four detection systems and the reference method are closer to 1 and 0 than before calibration. CONCLUSIONS: The precision, reference interval, and linear evaluation of the four detection systems are good. The ID-LC-MS/MS reference method assigning fresh frozen serum samples as calibrators can improve the accuracy and comparability of the results of different detection systems.

Optimization of an enzyme-coupling method by spectrophotometer for serum adenosine deaminase: As a candidate reference method.

Adenosine deaminase (ADA) is a key enzyme of adenosine metabolism. There are currently various kits and systems available for ADA measurement, and all yield variable results. This study optimized a reference measurement procedure (RMP) for serum ADA for the standardization of routine methods. ADA coupled with purine-nucleoside-phosphorylase, xanthine-oxidase and peroxidase was selected as the basic method and was optimized using Response Surface Methodology. Then the performance was validated and the results were compared after replication by 3 other reference laboratories. A reference interval was also developed. In addition, this optimized method was applied to calibrate a routine system. The intra-assay precision was 0.44% at both concentrations of 29.8 and 100.4 U/L, and inter-assay precision was 1.01% and 0.95% at 30.1 and 100.3 U/L, respectively. The linearity was up to 351.9 U/L (R2 = 0.9998), with no significant interference or carryover (<5%). A Comparison among 4 reference laboratories showed good reproducibility (R2 ≥ 0.9975). The procedure proved valid for a reference interval of 11.7-38.5 U/L. The mean relative deviation for a routine system was -55.9% and -3.7% before and after calibration. This candidate RMP for serum ADA can potentially be used for standardization of clinical systems.

Evaluation of a bracketing calibration-based isotope dilution liquid chromatography-tandem mass spectrometry candidate reference measurement procedure for 17α-hydroxyprogesterone in human plasma.

A candidate reference measurement procedure (RMP) based on isotope dilution coupled with liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) was developed and validated for the quantification of 17α-hydroxyprogesterone (17-OHP) in human plasma. 17-OHP spiked with a deuterium-labeled internal standard was extracted from plasma by liquid-liquid extraction with 1 mL n-hexane/ethyl acetate (3:2, v/v). Reversed-phase chromatography and positive electrospray ionization were used in the ID-LC-MS/MS. Gradient elution coupled with use of a C18-packed ultrahigh-performance liquid chromatography column allowed complete baseline resolution of 17-OHP from its structural analogue desoxycorticosterone in 6 min. To determine the 17-OHP level in human plasma, a bracketing calibration method was used to give higher accuracy and precision. The limit of detection and the lower limit of the measuring interval for the candidate RMP were 2.1 pg/mL (6.4 pmol/L) and 4.6 pg/mL (13.9 pmol/L), respectively. Extraction recovery was determined to be (96.08 ± 3.03)% (n = 3). Imprecision (intra-assay and interassay) was 4.03% or less at 0.83, 15.19, 64.22, and 313.46 ng/mL (2.51, 45.97, 194.34, and 948.56 nmol/L, respectively). Recoveries ranged from 98.05% to 102.24%. When comparing our RMP results with those obtained with an established RMP via International Federation of Clinical Chemistry and Laboratory Medicine external quality assessment scheme for reference laboratories in laboratory medicine (RELA) samples, we found that the biases ranged from -1.99% to 3.08% against the targets. No interference was observed, and the linear response ranged from 0.47 to 958.63 ng/mL (1.42 to 2900.90 nmol/L). Moreover, the candidate RMP was used to measure the concentration of 17-OHP in human plasma and was compared with an immunoassay using 40 plasma samples. The performance of the method meets the needs of an RMP (total coefficient of variation of 5% or less and bias of 3.08% or less). This method can be used for reference material value assignment of 17-OHP in human plasma matrix. It could also serve as an accurate reference baseline for routine methods to increase the accuracy and precision of certain clinical laboratory measurements. Graphical abstract Selected ion chromatograms obtained by liquid chromatography-tandem mass spectrometry with a C18 column for 17α-hydroxyprogesterone (17-OHP) from a plasma sample.

A Pre-Analytical Performance Evaluation for Measurement of Serum Creatinine in a Multicenter Clinical Trial Study.

BACKGROUND: Our multicenter clinical trial study for stage 4 chronic kidney disease (CKD) populations was conducted at 21 centers in China during the period 2011 to 2016. The CKD definition is based on glomerular filtration rate (GFR) values which can be estimated by creatinine-based predictive formulas. The validity and reliability of GFR estimation is thus largely dependent on the accurate and precise serum creatinine (SCr) measurements. As an integral part of this multicenter study, it is important to ensure the precision, accuracy, and center-to-center comparability of the SCr results. METHODS: Prior to initiating the study, we unified the measurement method of SCr determination as an enzymatic method and standardized the procedure in all of the laboratories. Then, the analytical performance of each analyzer at each laboratory was evaluated, including precision, accuracy, and comparability. RESULTS: All within-run and total CVs of the low and high level internal quality control (IQC) were comprised between 0.2% and 4.1% (< 1/3 CLIA'88). Total error of the IQC fall within the maximum 12% at all centers. The analytical bias against the Standard Reference material 967a target was less than ± 0.5% at Central Laboratory, indicating good accuracy. Correlation between the analyzers and the reference method were very high (r > 0.99). Passing-Bablok regression showed no significant deviation from linearity (p > 0.05). Bland-Altman analysis also showed good agreement (≥ 95% of results fell within the 95% limits of agreement). CONCLUSIONS: Performance evaluation helped in addressing preanalytical variations in measurement and gave op-timal quality assurance of laboratory measurement in the context of a multicenter clinical trial study.

Measurement of human serum unconjugated estriol without derivatization using liquid chromatography-tandem mass spectrometry candidate reference method and compared with two immunoassays.

A candidate reference measurement procedure (RMP) for measurement of unconjugated estriol in human serum has been developed and validated. The proposed method is highly reliable and uses isotope dilution coupled with liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) and requires no derivatization. An appropriate amount of serum was accurately weighed and spiked with an isotopically labeled internal standard. Unconjugated estriol and its internal standard were extracted from serum matrix using liquid-liquid extraction prior to reversed-phase LC-MS/MS. Calibrator bracketing was used to give higher specificity and accuracy for assigning serum level. The accuracy of the candidate RMP was validated by split-sample comparison to established RMPs. The lowest limit of detection (LLoD) and lowest limit of quantification (LLoQ) for developed RMP was estimated to be 0.14 nmol/L and 0.35 nmol/L, respectively. Both intra- and inter-assay imprecisions were ≤2.19% at 1.39, 17.34 and 69.35 nmol/L, respectively. Recoveries were 98.54% to 100.34% and linear response ranged from 0.35 to 173.38 nmol/L. No interference was observed. Biases were 5.6% and 2.8% against the targets of RELA2015A (3.87 nmol/L) and RELA2015B (40.62 nmol/L), respectively. Moreover, the candidate RMP was successfully applied to measure level of unconjugated estriol in serum samples of pregnant women (n = 3) and compared with two immunoassays in clinical laboratory. Our developed method is simple, accurate, and can be used as a candidate RMP to determine total unconjugated estriol level in human serum. Further improvement of certain immunoassays in accuracy and precision is needed. Graphical abstract Selected ion chromatograms by LC-MS/MS using a C18 column for uE3 from a serum sample.

Interference of Thalassemias on Hemoglobin A1c Measurement by Ion-Exchange High-Performance Liquid Chromatography Method Tosoh HLC-723 G8.

BACKGROUND: The presence of hemoglobinopathies could interfere with some assays for Hemoglobin A1c (HbA1c) measurement; therefore, the effect of thalassemia on ion-exchange high-performance liquid chromatography (IEHPLC) method Tosoh HLC-723 G8 (Tosoh G8) was evaluated. METHODS: A total of 43 normal controls and 101 thalassemia patients were quantified by Premier Hb9210 and Tosoh G8 (variant-mode) systems. At the same time, 7 normal controls and 8 thalassemia patients were confirmed by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) reference method for verification. RESULTS: For normal controls, the HbA1c values of Tosoh G8 system (y) showed great correlation and agreement with those from Premier Hb9210 (x) (y = 0.9688x + 0.2151, r = 0.9951; mean difference 0.02 ± 0.30%), and no significant relative bias above 7% was observed; the HbA1c values obtained by Tosoh G8 were consistent with the IFCC targets (relative bias < ± 6%) in all of the samples. However, for thalassemia, the correlation between Tosoh G8 (y) and Premier Hb9210 (x) became relatively low (y = 0.8079x + 1.2897, r = 0.7780); the HbA1c values of 91.1% of the samples (92/101) obtained by Tosoh G8 were higher than those by Premier Hb9210 (mean difference 0.33 ± 0.48%) and a significant positive bias above 7% was noticed in 43.3% (45/101) thalassemia patients; when compared with the IFCC targets, the 87.5% (7/8) relative bias was > ± 6%. CONCLUSIONS: Thalassemia could directly affect the measurement of HbA1c using the IE-HPLC method Tosoh G8 and the clinical laboratorial staff should pay close attention.

Simultaneous quantitation of endogenous estrone, 17ß-estradiol, and estriol in human serum by isotope-dilution liquid chromatography-tandem mass spectrometry for clinical laboratory applications.

Estrogen measurements are important in the assessment of female reproductive function and have expanding roles in other fields. A simple, accurate, highly sensitive and specific isotope-dilution liquid chromatography-tandem mass spectrometry method was developed and evaluated to simultaneously measure three endogenous estrogens in serum: estrone (E1), 17ß-estradiol (E2), and estriol (E3). Chromatographic separation was achieved on a C18 column before electrospray ionization triple-quadrupole mass spectrometry in multiple reaction monitoring mode. The sample preparation in this assay requires no derivatization and extraction by liquid-liquid extraction. After optimization of the extraction conditions, the final extraction efficiency of E1, E2, and E3 was 83.8%, 78.9%, and 77.3% respectively. The metabolites and structural analogs that have the same molecular masses as the estrogens were separated under the optimized liquid chromatography conditions. Method validation showed satisfactory linearity over the concentration range of 20-10000 pg mL-1 for all three estrogens (r 2 > 0.997). The limits of quantification were 5, 10, and 10 pg mL-1 for E1, E2, and E3 respectively, and their recoveries ranged from 94.7% to 103.5%. The accuracy of the proposed method was further evaluated with use of certified reference materials BCR-576, BCR-577, and BCR-578 for E2 and 2014 International Federation of Clinical Chemistry and Laboratory Medicine External Quality Assessment Scheme for Reference Laboratories in Laboratory Medicine samples for E3, whose certified values were determined by reference methods. Great agreement was observed between the measured values and the certified values. Satisfactory precision (coefficients of variation less than 7.44%) was also obtained for the three estrogens. Moreover, the proposed method was successfully applied to measure the three estrogens in serum samples of pregnant women in the second trimester and to assess the accuracy of chemiluminescent immunoassays in clinical laboratories by determination of E2 and unconjugated E3 in serum samples. Graphical Abstract Schematic representation of the simultaneous quantitation of three major endogenous estrogens in human serum by ID-LC-MS/MS.

Clinical Correlates and Reference Intervals for Cystatin C in a Han Population from Southeast China.

BACKGROUND: Cystatin C (CysC) is an endogenous filtration marker for estimation of kidney function. This study aimed to define the reference interval (RI) for serum CysC in a southeast Chinese adult population and to explore the variables that affect serum CysC levels. METHODS: 532 reference individuals (259 male, 273 female, aged 18 - 79 years) were recruited from Guangzhou, China. Multiple regression analysis (MRA) was used to investigate the association between serum CysC levels and clinical factors including age, gender, body mass index, lifestyle, and biochemistry parameters. Reference values were defined using a parametric method according to Clinical and Laboratory Standards Institute guideline (C28A3). RESULTS: The mean serum CysC levels were significantly lower in females than in males (p < 0.001). Serum CysC levels increased with age (~0.047 mg/L increase per decade). MRA demonstrated that serum CysC levels correlated significantly with serum creatinine (Cr), high density lipoprotein (HDL-C), alkaline phosphatase (ALP), albumin (ALB), and uric acid (UA) concentrations, although their relationships were less prominent than those of gender or age. The RIs for serum CysC levels were calculated at 0.73 - 1.17 mg/L for subjects aged 18 - 49 years and at 0.73 - 1.49 mg/L for those aged 50 - 79 years. CONCLUSIONS: The RIs for serum CysC were established in a southeast Chinese population. In addition to gender and age, serum Cr, HDL-C, ALP, ALB, and UA also influenced serum CysC levels.

Development of reference intervals for serum alkaline phosphatase among adults in Southern China traced to the new IFCC reference measurement procedure.

BACKGROUND: Serum alkaline phosphatase (ALP) plays a critical role in the diagnosis of various diseases, and the establishment of relevant, reliable reference intervals (RI) is key to avoiding misdiagnoses. In 2011, IFCC published the new reference measurement procedure (RMP) for the determination of serum ALP in which one of the main modifications was the measuring temperature of the assay. Here, the new RMP was used to help establish RIs for serum ALP concentrations in healthy Chinese Han. METHODS: Volunteer individuals in Guangdong province, China (n=1622) were screened by questionnaire and laboratory testing for eligibility as a reference. Blood (20 mL) was collected and samples were measured by the Roche Modular system using the new RMP for the serum ALP compatible method. Partitioning of values by gender and/or age was evaluated with a standard normal deviate test after removing outliers. A simple non-parametric method for a two-sided 95% distribution of reference values was calculated. RESULTS: Serum ALP concentrations were obtained from the cohort of eligible reference individuals (n=658). The RI for serum ALP in males age 18-79 years was 48-131 U/L. Females were partitioned into two age groups based on statistical analysis, 18-49 years and 50-79 years, and the RIs derived were 40-106 U/L and 57-159 U/L, respectively. CONCLUSIONS: RIs for serum ALP for Chinese Han individuals in between the ages of 18 and 79 years were determined and required partitioning due to the higher ALP values of females age 50-79 years.

Matrix Effects in Proficiency Testing Materials Influence the Accurate Measurement of Gamma-Glutamyltransferase Activity.

BACKGROUND: A consensus on an accurate method to measure γ-glutamyltransferase (GGT) activity for clinical purposes has not been achieved among practicing clinical laboratories. To improve analytical trueness, we evaluated the influences of matrix effects in proficiency testing (PT) materials on the measurement of GGT activity in human serum samples. METHODS: Five fresh frozen human samples (FFS1-5) and five lyophilized proficiency testing materials (Lyo1-5) were distributed to 23 participating clinical laboratories for the measurement of GGT activity. Target GGT activity values for the samples were obtained by using previously approved reference methods. The results obtained by the Beckman Coulter Unicel DxC 800 Synchron analyzer were compared to the target values assigned by two reference laboratories, and the commutability of the lyophilized materials was evaluated according to Clinical and Laboratory Standards Institute (CLSI) guideline EP14-A2. RESULTS: The relative bias between the results obtained by the Beckman Coulter analyzer and the reference target values ranged from -27.2% to -18.0% for FFS1-5 and from 9.1% to 2.5% for Lyo1-5. Non-commutability of all lyophilized samples falling outside of the 95% prediction interval was observed. CONCLUSIONS: The results obtained for the lyophilized PT materials were deemed acceptable within the total allowable errors, suggesting that matrix effects may impart a false sense of confidence that clinical analytical systems are performing very well. A primary reference measurement procedure on fresh frozen serum provides a valuable method for evaluating the trueness of results measured by PT.

Application of New Reference Procedure for ALP Measurement in the Clinical Laboratory.

BACKGROUND: Alkaline phosphatase (ALP) is routinely analyzed in clinical laboratories for the comprehensive assessment of hepatic and osteal diseases. The official reference measurement procedure (RMP) of ALP was released by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) in 2011. However, most of the commercial kits still trace back to the old version (1983). There was a difference between the trace measurement procedure and the RMP. Therefore, the discrepancy among clinical systems and application of the new RMP for ALP in clinical laboratories was studied. METHODS: According to the recommendation of the IFCC, the RMP for ALP (2011) was reproduced. The reference measurement system (RMS) for serum ALP and 19 clinical systems were included in the external quality assessment (EQA). The relative bias was calculated between the clinical systems' results and RMS, as well as each clinical system and the average value of all clinical systems. The qualified rates (passing score in percentage) for the 19 clinical systems were compared by using two different standards. In the comparison experiment, two clinical systems were evaluated before and after calibration by RMP. The clinical acceptability at the medical decision point was evaluated. RESULTS: The performance of the reproduced RMP for ALP was: total imprecision was 0.33% and 0.42% at 336.9 U/L and 138.74 U/L, respectively. The accuracy was in the acceptable range. Excellent linearity was obtained for linear regression (R = 0.9998). In the EQA experiment, the relative bias of clinical systems and RMP ranged from -26.36% to 19.49%, and the majority of them had a negative value. Relative bias of clinical systems and the average value of 19 clinical systems ranged from -24.28% to 33.48%. The qualified rate for clinical systems was 53% - 89% evaluated by Standard 1 and was 95% - 100% evaluated by Standard 2. In the comparison experiment, the relative bias for the two clinical systems decreased and both of the clinical systems showed less relative bias at the medical decision points after calibration by RMP. CONCLUSIONS: There was a much higher discrepancy among clinical systems for the testing of serum ALP. Traceability and standardization would likely be improved for clinical systems by the application of RMP for ALP (2011) in clinical laboratories.

Effect of CD95 on inflammatory response in rheumatoid arthritis fibroblast-like synoviocytes.

OBJECTIVE: Many CD95-expressing cells don't always undergo apoptosis after stimulation with CD95 ligation. The purpose of this paper is to investigate the role of expression of CD95 (Fas/Apo1) on inflammatory response in fibroblast-like synoviocytes (FLS) obtained from rheumatoid arthritis (RA) and to evaluate the role of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB or Akt) pathways within this process. METHODS: The expression levels of CD95 were monitored by immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR). Apoptotic cells were detected by in situ apoptosis detection (TUNEL) assay. The RA-FLS were treated with agonistic anti-CD95 antibody or CD95 siRNA. Then the proliferation was detected by CCK-8, and mRNA level of inflammatory cytokines was detected by RT-PCR. After the RA-FLS were treated with agonistic anti-CD95 antibody, the total Akt and pAkt protein expression was analyzed by Western blot, and the changes mentioned above were observed while pre-incubated with the PI3K inhibitor LY294002. RESULTS: A significant increase of CD95 antigen was found in RA compared with osteoarthritis (OA) samples, while apoptosis in RA synovial tissue was not obvious. Low concentrations of agonistic anti-CD95 antibody could promote RA-FLS growth and interleukin-6 (IL-6) mRNA expression, while high concentrations could induce apoptosis. And both of these phenomena could be inhibited by CD95 siRNA. Agonistic anti-CD95 antibody could stimulate the expression of pAkt, and PI3K specific inhibitor LY294002 could induce opposite changes. CONCLUSION: Stimulation of CD95 could promote RA-FLS proliferation and inflammation, and activation of the PI3K/Akt signaling pathway might be the possible mechanism.

[Triptolide inhibites Th17 cell differentiation via regulating cyclooxygenase-2/ prostaglandin E2 axis in synovial fibroblasts from rheumatoid arthritis].

Triptolide (TPT), an active compound extracted from Chinese herb Tripterygium wilfordii , has been used in therapy of rheumatoid arthritis (RA). In this study, after synovial fibroblasts from rheumatoid arthritis (RASFs) were treated with TPT, we investigated its effect on the differentiation of Th17 cells. Firstly, the mRNA level of cyclooxygenase (COX) wad detected by qRT-PCR and the protein level of prostaglandin E2 (PGE2) was tested by ELISA in RASFs treated with different concentrations (0, 10, 50, 100 nmol L-1 ) of TPT. Then after TPT pre-treated RASFs and RA CD4 + T cells wer e co-cultured for 3 days in the presence or absence of PGE2, IL-17 and IFN-gamma production in CD4 T cell subsets were detected by flow cytometry. The results showed TPT decreased the mRNA experssion of COX2 and the secretion of PGE2 in RASFs in a dose-dependent manner(P <0. 05). We further found that differentiation of Thl7 cells was downregulated in a dose-dependent manner, and exogenous PGE2 could reverse the inhibition of Th17 cell differentiation(P <0. 05). Taken together, our results demonstrated that TPT inhibited the mRNA level of COX2 and the secretion of PGE2 in RASFs, which partly led to impaired Th17 cell differentiation in vitro.

A Method for Production of Human Adenosine Deaminase 1 (ADA1) in Pichia pastoris Provides Needed Quality Control Material for Clinical Laboratories.

OBJECTIVE: Adenosine deaminase (ADA) plays a major role in maintaining metabolic homeostasis via catalysis of hydrolytic deamination of adenosine to inosine. The ADA1 isoenzyme of ADA is an analyte tested in clinical laboratories; however, lack of quality control (QC) material in terms of enzyme homogeneity, stability, and coverage of the clinically relevant analytical measurement range (AMR), poses a challenge for adequate monitoring of this analyte. The aim of this study was to address the need for manufacture of QC material through recombinant expression of catalytically active ADA1 in eukaryotic cells (Pichia pastoris GS115). METHODS: The coding region of ADA1 gene was amplified by PCR and ligated into plasmid pPICZαA, followed by transfer into P. pastoris using electroporation. Recombinant ADA1 produced by P. pastoris was purified using a Ni-NTA resin column, yielding 5 mL of purified ADA1 with an activity of 4200.6 U/L. Purified ADA1 protein was added to human donor serum as the appropriate matrix for QC materials preparation. RESULTS: One hundred vials of lyophilized ADA1 were prepared at clinically significant concentrations at 41.6 U/L and 115.5 U/L (50 vials each). Both concentrations were homogenous and stable at room temperature (RT, 22-24°C) for at least 7 d, at 4°C for 3 months, and at -20°C for 12 months. Reconstituted aliquots of QC material were found to be stable at -20°C for up to 60 d and should be used within 8 h or 48 h when stored at RT or 4°C, respectively. CONCLUSION: Success of this ADA1 expression system presents a potential solution to increase production options available to clinical laboratories.

Simultaneous quantitation of 17 endogenous adrenal corticosteroid hormones in human plasma by UHPLC-MS/MS and their application in congenital adrenal hyperplasia screening.

Accurate investigation of adrenal hormone levels plays a vital role in pediatric endocrinology for the detection of steroid-related disorders. This study aims to develop a straightforward, sensitive UHPLC-MS/MS method to quantify 17 endogenous adrenal corticosteroid hormones in human plasma. These hormones are the main ingredients in the synthetic and metabolic pathways of adrenal corticosteroid hormones. Chromatographic separation was achieved on a C18 column before electrospray ionization triple-quadrupole mass spectrometry in multiple reaction monitoring mode with a run time of 7 min. The samples were extracted by liquid-liquid extraction and required no derivatization. Analytical performance was evaluated, including linearity, analytical sensitivity, accuracy, precision, and specificity. Plasma specimens from 32 congenital adrenal hyperplasia (CAH) patients and 30 healthy volunteers were analyzed to further reveal the diagnostic value of multiple steroid hormones in the synthetic and metabolic pathways of adrenal corticosteroid in CAH diagnosis. All hormones were effectively extracted and separated using our method. The method was essentially free from potential interference of isomers or structural analogues. The imprecisions were <10%. The lower limits of quantification varied from 0.05 to 15.0 ng/ml. Good linearity coefficients (r 2 > 0.998) were also obtained for most hormones in the required concentration range, except for 21-deoxycortisol (r 2 = 0.9967) and androstenediol (r 2 = 0.9952). The recoveries for the steroid hormones ranged from 91.7 to 109.8%. We developed the UHPLC-MS/MS method for the simultaneous measurement of steroid hormones. The results showed that measurement of steroid hormones simultaneously could improve the diagnostic efficiency of CAH.

Determination of serum 25-hydroxyvitamin D status among population in southern China by a high accuracy LC-MS/MS method traced to reference measurement procedure.

OBJECTIVE: We aimed to describe the 25-hydroxyvitamin D (25(OH)D) status of southern Chinese individuals by a high-accuracy liquid chromatography tandem mass spectrometry (LC-MS/MS) method which can trace to reference measurement procedure. MATERIALS AND METHODS: From January 2018 to June 2019, a total of 4775 southern Chinese individuals were evaluated in our study. The serum levels of parathyroid hormone (PTH) were detected simultaneously in 162 cases. 25(OH)D was determined by LC-MS/MS, and PTH was detected using routine automated analysers. The distribution of the concentration, prevalence and seasonal variability of 25(OH)D in males and females of different age groups were studied. RESULTS: The mean 25(OH)D concentration in our study was 32.57 ng/mL (4.20-101.40 ng/mL). The global 25(OH)D concentration in males was higher than that in females of different age group. The prevalence of vitamin D deficiency (< 20 ng/mL) in females (16.65%) was higher than that in males (6.83%). The prevalence of vitamin D deficiency (< 20 ng/mL) was most common in winter (22.98% of all women and 15.49% of all men). 25(OH)D concentrations were higher in those from whom blood samples were collected in summer and autumn than in winter and spring. 25(OH)D2 was detected in 672 serum samples (14.07%). In addition, there was a negative correlation between the concentrations of 25(OH)D and serum PTH (r = - 0.149, P < 0.05). CONCLUSION: Our study demonstrated that the average serum 25(OH)D concentration in southern Chinese individuals was higher than that in other Chinese cohorts by a high-accuracy LC-MS/MS method. The global 25(OH)D concentration in males was higher than that in females of different ages, and the prevalence of vitamin D deficiency in females was higher than that in males. Seasonal change was an important aspect of 25(OH)D concentration in young and middle-aged people but became less relevant for that in older subjects. 25(OH)D2 detection was of minor practical significance in our study. In addition, we also found that there was a negative correlation between the serum levels of 25(OH)D and PTH in southern Chinese individuals.

Selenepezil, a Selenium-Containing Compound, Exerts Neuroprotective Effect via Modulation of the Keap1-Nrf2-ARE Pathway and Attenuates Aß-Induced Cognitive Impairment in Vivo.

Oxidative stress is a major risk factor for neurodegenerative disease. The Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid 2 related factor 2 (Nrf2)-antioxidant response element (ARE) pathway is one of the most potent defensive systems against oxidative stress. Selenepezil, a selenium-based compound, was previously found to exhibit excellent acetylcholinesterase (AChE) inhibition, to mimic endogenous glutathione peroxidase (GPx) activity, and to exhibit scavenging activity for hydrogen peroxide in vitro. However, none of these activities have been evaluated in a cellular model, and detailed molecular mechanisms are not elucidated. Moreover, whether selenepezil ameliorates memory deficits in vivo remains unknown. This study validated the cytoprotective effect of selenepezil against 6-hydroxydopamine (6-OHDA)- or H2O2-induced SH-SY5Y cell damage via alleviation or neutralization of intracellular microtubule disorder, reactive oxygen species (ROS) accumulation, mitochondrial dysfunction, and cell apoptosis. Our study clearly demonstrated that selenepezil pretreatment exhibited remarkable cytoprotective effect in a Nrf2-dependent manner via activating the Keap1-Nrf2-ARE pathway and stimulating the transcription of Nrf2-ARE-regulated cytoprotective genes. Moreover, selenepezil·HCl exerts neuroprotective effect via attenuating Aß-induced cognitive impairment in Alzheimer's disease (AD) rat and was more active than the reference drug donepezil. In summary, selenepezil deserves further consideration for AD therapy.