Objective To study the effects of citrullinated vimentin (cVim) on the maturation and immunologic function of dendritic cells (DCs) from rheumatoid arthritis (RA) peripheral blood.Methods In the present study,mononuclear cells were isolated from the peripheral blood of patients with RA and cultivated in media containing GM-CSF and IL-4 to generate immature DCs (imDCs).The imDCs generated were stimulated with citrullinated vimentin and vimentin.LPS was used as the positive control and PBS was used as the negative control.The expression of surface molecules on the DCs,such as CD14,CD80,CD83,CD86,MHC Ⅰ and MHC Ⅱ were analyzed with FACS.The capability of the stimulatory activity of the DCs on allogeneic T cells in mixed reaction was tested by MTS.t-test was used for statistical analysis.Results Compared to untreated DCs,DCs treated with LPS increased the expression levels of MHC Ⅱ,CD80,CD83 and CD86 (1.07±0.14,1.25±0.13,1.90±1.08,2.44±0.65,P＜0.05),while cVim increased the expression levels of MHC Ⅱ ( 1.18±0.09,P＜0.05) and CD83 ( 1.97±0.99,P＜0.01 ),and Vim decreased the expression levels of CD80 (0.82±0.18,P＜0.01 ).It was demonstrated that the expression levels of MHC Ⅱ on DCs pulsed with cVim were significantly higher than that of the DCs with LPS,but the expression levels of CD80 and CD86 were not significantly different.The expression levels of MHC Ⅱ and CD83 on DCs pulsed with cVim were significantly higher than that of the DCs with Vim.The mixed lymphocyte reaction showed that the DCs induced by LPS and cVim trigerred the proli-feration of allogenic T cells obviously.Conclusion This result suggests that cVim could promote the phenotypic maturation of DCs and increase the expression of costimulatory molecules.

Objective To investigate the clinical value of pregnancy-associated plasma protein A (PAPP-A) in patients with acute coronary syndrome (ACS).Methods The study subjects comprised of 249 patients with ACS [ 50 patients with ST elevation acute myocardial infarction( STEMI),43 patients with non-ST elevation acute myocardial infarction (NSTEMI), 156 patients with unstable angina pectoris (UAP) ] from July 2008 to December 2009 at Shanghai East Hospital affiliated to Tongji University.The patients were divided into single-vessel lesions group,double-vcssel lesions group and three-vessel lesions group according to the coronary artery stenosis.A group of 205 healthy subjects admitted for health physical examination were used as conuols.Blood samples were collected from ACS patients and control subjects.Serum PAPP-A,creatine kinaseisoenzyme MB (CK-MB),high-sensitivity troponin-T (hs-TnT) were measured by clectrochemiluminesence and high-sensitivity C-reactive protein (hs-CRP) was measured by immune scatter turbidity method.Analysis of variance ANOVA and Kruskal-Wallis H test were used for statistical analysis.Results Serum PAPP-A in the STEMI,NSTEMI,UAP and normal control group were 10.45(5.54 - 16.08),6.56(4.68 - 9.55),5.70(4.12 - 8.50),5.23 (4.00 - 5.88) mIU/L,respectively,and the difference was statistically significant (H =43.94,P ＜ 0.01 ).Pairwise comparison showed that PAPP-A in STEMI,NSTEMI,UAP were significantly higher than the healthy control group and differences were statistically significant ( t =6.80,2.46,1.72,P ＜ 0.05 ).The PAPP-A had a sensitivity of 52.2％ and specificity of 80％ in diagnosis of ACS.The positive rate of PAPP-A was 44.5％ (69/155) in patients with negative hs-TnT.The serum levels of PAPP-A in the three-vessel lesions group [7.72(5.03 -12.46) mIU/L] was higher than that in the single,double groups [ 5.35 ( 4.14 - 8.59 ),6.05 (4.42 -9.58 ) mIU/L ],and the difference had statistical significance( t =2.00,1.57,P ＜ 0.05 ).There was obvious correlation between the level of serum PAPP-A and the level of hs-CRP ( r =0.524,P ＜ 0.05 ),and there was weak correlation between the PAPPA and CK-MB or hs-TnT (r=0.326,0.343,P＜0.05).Conclusions The results shows that PAPP-A is elevated in patients with ACS.It may be used as a serum biomarker for vulnerable plaques in patients with ACS and has a clinical value for ACS diagnosis especially in patients with negative hs-TnT.

Objective To establish an ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for detecting α-hydroxybutyrate (α-HB) in serum.Methods Electrospray ionization negative ion and multiple reaction monitoring mode were used to detect serum α-HB.The linearity,low limits of quantification,precision,recovery and interference of UHPLC-MS/MS were evaluated.The reference interval of this method was established in 130 serum samples (62 males and 68 females) from Shanghai East Hospital.Dixon method was used to judge the outliers and K-S test was used to analyze the data normality.The standard curve was scored by linear regression analysis.Results The total run time was 4 min of UHPLC-MS/MS method for the determination of α-HB.It has a good linear relationship in the range of 0.5-40.0 mg/L(r=0.999 4);the low limit of quantification was 0.5 mg/L;the in-batch and inter-batch coefficient of variation precision were less than 4.1％ and 6.3％,respectively;the recovery ranged between 95.8％ and 103.8％.Hemolytic samples (about 5 g/L hemoglobin),lipemic samples (about 12 mmol/L triglyceride),icteric samples (about 150 μmol/L total bilirubin) had no significant interference to the detection.The reference range of the apparent healthy population was 1.46-6.48 mg/L.Conclusions A method for the determination of serum α-HB by UHPLC-MS/MS was established.The method was simple,rapid,and could be used for the detection of clinical samples.

The establishment of laboratory medicine reference system is an important way for routine detection results to achieve traceability.The complete reference system consists of reference measurement procedures,reference materials and reference laboratories.The reference measurement procedure is usually characterized by a thorough study with a small measurement uncertainty.Based on the advantages of high specificity and high sensitivity in quantitative analysis, mass spectrometry plays an important role in the research of the reference measurement procedures in laboratory medicine.In the past 40 years, mass spectrometry technology has been more and more widely applied in medicine, electrolytes, metabolites and substrates, non-electrolyte metals, non-peptide hormones, protein and vitamin.It provides a powerful guarantee for the precise detection of these well-defined measureds.

Objective:To determine the analytical performance of a candidate reference measurement procedure for 17α-hydroxyprogesterone in human serum by liquid chromatography-tandem mass spectrometry (LC-MS/MS).Methods:The serum spiked with a deuterium-labeled internal standard was extracted from serum from individual undergoing physical examination by liquid-liquid extraction with n-hexane/ethyl acetate (3∶2, v/v), separated by C18 reversed-phase chromatography and detected by positive electrospray ionization mass spectrometry. According to the Clinical and Laboratory Standards Institute (CLSI) C62-A documents, the analytical performance including linearity, limit of detection,limit of quantitation,relative matrix effect,precision and trueness,carry-over and specificity was evaluated.Results:The linear range of 17α-hydroxyprogesterone by LC-MS/MS was 0.21-119.67 μg/L. The limit of detection and limit of quantitation were 5.218 ng/L and 0.116 μg/L. The relative matrix effects were -0.02%, -0.40% and -0.90% for sera and solution mixtures in 3 different ratios (50∶50, 80:20 and 20∶80). The coefficients of variation ( CVs) of intra-assay were 1.73%-2.11%, 0.98%-1.71%, 0.47%-0.87% at 0.164 μg/L, 14.81 μg/L, 81.63 μg/L and the CVs of inter-assay were 1.82%, 1.03%, 0.80% at above three concentrations. The average recovery rates of 3 levels (0.5, 20 and 100 μg/L) were 100.4%, 101.7%, 102.8%, respectively. The measured values of GBW09829 of National Institute of Metrology were within the specified uncertainty range. Conclusion:The candidate reference measurement procedure for 17α-hydroxyprogesterone in human by LC-MS/MS is established with good accuracy and precision, which can be clinically used for measurement traceability.

Objective To establish an isotope dilution liquid chromatography tandem mass spectrometry method (ID-LC-MS) for quantification of serum apolipoprotein E and phenotyping. Methods Method establishment. Samples underwent denaturing, alkylation and trypsin digestion with addition of internal standards as isotope labelling arginine. SB-C18 column was used for the liquid chromatographic separation and mass spectrometry positive ion mode and multiple reaction monitoring were employed for quantification and phenotyping. Precision, accuracy and linearity were investigated for method evaluation. 40 serum samples from Shanghai Dongfang Hospital during Oct. to Dec., 2018 were used for method comparison between ID-LC-MS and immunoassay. Deming regression and Bland-Altman were used for method comparison analysis and SPSS 24 for linearity. Results Target peptides reached their releasing maximum within 4 hours and SE did at 3 hours. 3 phenotyping of ApoE were observed, such as E3/E3, E2/E3 and E3 / E4. The imprecision of IQC was 5.2 ％ . The relative bias for low and high levels of accuracy-based samples was 7.6 ％ and 3.6 ％, respectively. Deming regression showed the intercept with 95 ％ confidence interval (CI) was 6.44-11.44 (P<0.05 and the 95％ confidence interval for the slopewas 0.77-0.89 (P<0.05). The coefficient was r=0.97. The mean difference was - 2.95 mg / L with 95 ％ CI-4.26--1.65 mg/L. The linearity covered from 16.9 to 58.5 mg/L. Conclusion ID-LC-MS can be used to quantify serum apolipoprotein E and simultaneously detect its phenotyping.

Objective: To establish a reference measurement procedure for the determination of human serum homocysteine by isotope dilution high performance liquid chromatography-tandem mass spectrometry (ID-LC/MS/MS), and to apply it to establish sample target values for external quality assessment (EQA) in clinical laboratories. Methods: The reference method of Hcyquantification in our laboratory was establishedaccording to the method recommended by the Joint Committee for Traceability in Laboratory Medicine (JCTLM). The precision, trueness, specificity, residue and matrix effect of the method were evaluated. The reference method was applied to establish Hcy target values for samples of the second EQA in Shanghai of 2018. Results: The method detects 12.5μmol/L and 37.4μmol/L samples in three batches in three days, and the CV between batches is 1.03% and 2.10%, respectively. The measured values of Standard reference material (SRM) 1955 of National Institute of Standards and Technology (NIST) were within the specified uncertainty range. No matrix effect and carryover were observed. The second EQA data in 2018 showed that the average value of domestic reagent group was lower than that of reference method, and that of imported reagent group was higher than that of reference method. Conclusion Thereference measurement procedure of ID-LC/MS/MS was successfully established to determine the human serum homocysteine. It is expected to play a role in tracing the quantities of Hcy in clinical laboratories.

Objective: To establish a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for the detection of serum oleic acid (OA), and preliminarily evaluate the role of OA in insulin resistance (IR) of type 2 diabetes (T2DM). Methods: OA-［ 13 C 5 ］ was used as isotope-labeled internal standard, and the ion pairs of OA and OA-［ 13 C 5 ］ were 281.3/281.3 and 286.3/286.3, respectively. The ultrapure water was used as mobile phase A and methanol: acetonitrile (1∶1, v/v) as mobile phase B in a ZORBAX SB-Aq C18 reversed phase column. Meanwhile, the gradient elution system with a flow rate of 0.3 mL/min was used. According to the CLSI guidelines (EP15-A3), the reliability of the established method was evaluated by detecting the performance indicators such as precision, trueness, linear range, stability and carrying contamination rate. Serum OA levels were detected by the established HPLC-MS/MS method in 109 patients with clinically diagnosed T2DM and 100 healthy controls. The insulin resistance index (HOMA-IR) was calculated to evaluate IR, and the relationship between OA and IR was further analyzed. Results: The established HPLC-MS/MS method for the detection of serum OA had good specificity and linearity in the range of 10-1 000 μmol/L (y=0.007 55x+0.004 83,r=0.997 7), and the low limit of quantification (LLOQ) was 10 μmol/L. It also had good precision, and the within-run coefficient of variation (CV) and total CV were not more than 1.62% and 1.73%, respectively, indicating that the method was suitable for the detection of serum OA. The serum OA levels in T2DM patients ［(425.58 ± 220.17) μmol/L］ were significantly higher than that in the healthy controls ［(113.20±58.00) μmol/L］, and serum OA levels were significantly correlated with HOMA-IR in T2DM patients and healthy controls. The area under the receiver operating characteristic (ROC) curve (AUC) of OA for the diagnosis of IR was 0.689. When the cut-off value identified by Youden index was 235.8 μmol/L, the sensitivity and specificity were 70.4% and 63%, respectively. When OA combined with fasting blood glucose (FBG) to diagnose IR, the AUC increased to 0.806, which was significantly higher than that of OA (P＜0.05). Conclusion A scientific and efficient HPLC-MS/MS method for the quantitative detection of serum OA is established successfully, which provides a reliable method for the dynamic monitoring of the changes of OA levels in the patients with metabolic diseases.