Objective To investigate the effect of measuring value transfer for human serum samples assigned by the reference laboratory network on improving the trueness of seven enzyme activities in clinical laboratories,such as ALT,AST,GGT,LDH,CK,AMY and ALP.Methods Depending on the medical imtitutions at all levels contacted by 5 reference laboratories in North China,South China,East China and Southwest China,the corresponding clinical laboratory measuring value transfer/traceability network was established.The frozen human serum samples with good interehangeability and standard material characteristics,including calibrator,sample 1 and sample 2,were provided by Beijing Aerospace General Hospital,and were assigned by 5 reference labotatories in four regiom.These samples were sent to 48 clinical laboratories.These clinical laboratories measured sample 1 and sample 2 according to their standard operating procedures,and then measured.the two samples again after adjusting their measurement system by using the supplied calibrator.The changes of trueness of detection results in these laboratories were evaluated according to the WS/T 403-2012 standard,and the changes of consistency for ALT and AST before and after measuring value tramfer were investigated.Results The results of AMY,ALP,GGT,CK and LDH calibrator,sample 1 and sample 2 assigned by the established network were 138.7 U/L,278.5 U/L and 68.3 U/L,265.3 U/L,94.5 U/L and 134.4 U/L,195.8 U/L,89.0 U/L and 158.9 U/L,393.7 U/L,260.0 U/L and 645.3 U/L,and 302.0 U/L,250.0 U/L and 452.7 U/L,respectively.The percentages of sample 1 and sample 2 which met the bias requirements of the WS/T 403-2012 standard before measuring value transfer for AMY,ALP and GGT were 65.9％ and 61.0％,76.6％ and 78.7％,and 66.7％ and 70.8％,respectively,while after measuring value transfer,they were 89.2％ and 83.8％,86.7％ and 80.0％,and 85.4％ and 91.7％,respectively.The percentages of sample 2 which met the bias requirements of the WS/T 403-2012 standard before measuring value transfer for CK and LDH were 64.6％ and 58.3％,respectively,while after measuring value trander,they were 93.5％ and 84.8％,respectively.The coefficients of variation (consistency) of sample 1 and sample 2 for ALT and AST before measuring value tramfer were 12.9％ and 11.3％,and 10.2％ and 8.9％,respectively,while after measuring value transfer,they were 9.3％ and 8.2％,and 5.6％ and 5.9％,respectively.Conclusion The calibration of routine measurement systems based on the measuring value transfer for human serum samples assigned by the reference laboratory network may improve the comparability of 7 enzyme actvities measurement results in chnical laboratories at all levels obviously,which deserves to be further spread.

Objective To investigate the expression levels of miRNA in kidney biopsies of child patients with nephrotic syndrome and the possibility of miRNA as potential markers in differentiating the pathologic subtypes of nephrosis.Methods Kidney biopsy specimens from 41 child patients with nephrotic syndrome,including 22 with nesangial proliferative glomeplonephritis (MsPGN),8 with minimal change disease (MCD) and 11 with endocapillary proliferative glomerulonephritis (ECPGN),were collected,and adult nephridial tissues from 8 patients without renal inadequacy were selected as controls.The expression levels of miR-191,miR-151-3p,miR-150,miR-30a-5p and miR-19b in nephridial tissues were detected by RT-qPCR,and their correlations with renal function related parameters were analyzed.Results Compared with the controls,the miR-191 levels in kidney tissues of child patients with nephrotic syndrome increased significantly (P ＜ 0.01),while the miR-151-3p levels decreased obviously (P ＜ 0.01).The expression levels of miR-150 in MCD patients were significantly lower than those in MsPGN and ECPGN patients and the controls (P ＜ 0.05).The expression levels of these miRNAs were positively correlated with serum IgG,TP and Cr levels,but negatively with serum TC levels (P ＜0.05).Conclusion The expression levels of miRNA in kidney tissues of child patients with nephrotic syndrome are related to pathological typing of nephrosis,and miR-150 may be a potential marker which may differentiate MCD from other subtypes of nephrosis.

Objective To establish a method of gold nanoprobe-based solution hybridization (GNBSH) to detect nucleic acid sequence-based amplification (NASBA) products for the rapid diagnosis of invasive aspergillosis (IA).Methods The Aspergillus specific 18S rRNA was amplified by NASBA and then the amplified products were hybridized with the gold nanoprobes which were modified with thiol compounds at the 5'end.Serum samples from 106 patients,including 14 with a definite IA,32 with suspected IA and 60 without IA,were detected by the established method,and the obtained results were compared with that of galactomannan (GM) test to evaluate its accuracy.Results The gold nanoprobes only hybridized with Aspergillus NASBA products but not other non-Aspergillus strains.The sensitivity,specificity and the area under the ROC curve (AUCROC) of the established GNBSH method for detecting 106 clinical samples were 82.61％ (38/46),81.67％ (49/60) and 0.890,respectively.The sensitivity,specificity and AUCROC of GM test were 56.52％ (26/46),83.33％ (50/60) and 0.723,respectively.Conclusion The established GNBSH method to detect Aspergillus NASBA products has high sensitivity and specificity and simple operation,which may be used to detect the infection of Aspergillus by clinical laboratories.

Objective To isolate and identify exosomes from serum samples of the patients with polymyositis / dermatomyositis (PM/ DM),and analyze their protein composition preliminarily.Methods Exosomes from serum samples of the patients with PM/DM were isolated and purified by the ExoQuickTM kit.The morphological characteristics and particle size of exosomes were determined by transmission electron microscope (TEM) and NanoSight analyzer,respectively.The surface markers of exosomes such as CD9,CD81 and Flotillin-2 were identified by western blot.The concentration and composition of exosome protein were determined by the BCA method and SDS-PAGE,respectively.Results The exosomes from serum samples of PM/DM patients displayed round or oval vesicles with membrane structure under TEM,and their diameter range was about (92 ± 67) nm.western blot showed that these exosomes expressed CD9,CD81 and Flotillin-2.The total protein concentrations of exosomes in the patients with PM/DM and healthy controls were 14.68 (6.00,32.55) μg/μL and 14.09 (8.00,23.28) μg/μL,respectively.SDS-PAGE showed that high-abundance proteins enriched in 55-70 kD in both PM/DM patients and healthy controls,and that there were different bands in 40-55 kD between them.Conclusion Exosomes are isolated from serum samples of the patients with PM/DM successfully,and their protein concentration and composition are analyzed preliminarily,which provides the experimental evidences for further finding differential proteins.

Objective To investigate the application value of interleukin-27 (IL-27) in the patients with acute coronary syndrome (ACS).Methods A total of 208 ACS patients were enrolled in the study,including 76 acute myocardial infarction (AMI) patients with ST elevation (STEMI),58 AMI patients with non-ST elevation (NSTEMI) and 74 unstable angina pectoris (UAP) patients.These patients were divided into single-vessel lesions,double-vessel lesions and three-vessel lesions groups,and 62 patients with chest pain syndrome (CPS) were selected as a control group.The plasma IL-27 levels of all patients were determined by enzyme linked immunosorbent assay (ELISA) and analyzed.Results The levels of plasma IL-27 (median[P25,P75]) in STEMI (308.64 [245.17,359.26] pg/mL),NSTEMI (256.88 [181.52,332.51] pg/mL) and UAP (218.12 [165.33,312.46] pg/mL) patients were significantly higher than that in CPS patients (100.66[68.98,228.86] pg/mL,P ＜ 0.01).The levels of plasma IL-27 in STEMI patients were significantly higher than that in NSTEMI and UAP patients (P ＜ 0.05).The positive rate of IL-27 in ACS patients with negative TnI was 54.24％ (32/59).The sensitivity and specificity of IL-27 in predicting ACS from chest pain patients were 80.29％and 58.06％,respectively.The levels of plasma IL-27 in the patients with three-vessel lesions were significantly higher than that with single-vessel lesions (P ＜ 0.05).Conclusion Plasma IL-27 levels in ACS patients increase obviously,which may be involved in the pathogenesis of ACS.IL-27 may be helpful for the diagnosis of ACS patients with negative TnI and the prediction of ACS state.

Objective To investigate the genotype and mutation frequency of thalassemia in child patients of Shenzhen region so as to provide evidences for the gene diagnosis and genetic counseling of thalassemia.Methods A total of 1 206 child patients suspected with thalassemia were retrospectively analyzed.The gene deletion of α-thalassemia was detected by Gap-PCR.The point mutations of α-thalassemia and β-thalassemia were determined by reverse dot blot(RDB)-PCR.The specimens suspected with HKαα and rare gene mutations were determined with nested PCR and gene sequencing,respectively.Results The detection rate of thalassemia was 76.9％ (927/ 1 206).Among them,α-thalassemia accounted for 40.5％ (489/1 206),and--SEA/αα was the most common gene mutation(75.1％);β-thalassemia accounted for 33.7％ (406/1 206),and the main IVS-2-654 (C→T) and CDM1-42 (-TCTT) heterozygous mutations accounted for 35％ and 32.5％,respectively.In addition,there were 32(2.7％) β-thalassemia patients with α-thalassemia mutation,1 patient with HKαα/ααQS,1 α-thalassemia patient with CD61 (AAG→TAG)/--SEA and 1 β-thalassemia patient with CD5 (CCT→C).Conclusion The are complicated gene mutation types and rare gene mutations of thalassemia in child patients of Shenzhen region.

Objective To analyze 4 child patients with 3-methylcrotonyl-coenzyme A carboxylase deficiency (MCCD) identified by neonatal screening and confirmed by urine gas chromatography-mass spectrometry (GC/MS) and genetic analysis.Methods Newborns whose C4DC + CSOH concentration was above 0.6 μmol/L in newborn screening were recalled for rescreening,and the CADC + C5OH concentrations in their mothers were detected.The child patients suspected with MCCD were further confirmed by urine GC/MS and genetic analysis.Results Three child patients were definitely diagnosed as MCCD by genetic analysis,including 1 MCCD,1 maternal MCCD and 1 paternal MCCD.The other 1 child patient suspected with MCCD had only one allele in MCCC1.Conclusion The mother and father of newborns with elevated C4DC + C5OH identified in neonatal screening should routinely perform MS / MS testing.When only one pathogenic locus is found in the suspected MCCD child patients by genetic analysis,they should be followed up regularly.

Objective To investigate the expression levels of serum LINC00978 in gastric cancer patients and its correlation with clinicopathological characteristics,and evaluate its clinical diagnostic value.Methods The expression levels of LINC00978 in gastric cancer cells and serum samples from the patients with gastric cancer or gastric benign diseases and healthy controls were detected by real-time qRT-PCR,and its correlations with clinicopathological parameters were analyzed.The diagnostic efficacy of serum LINC00978 was evaluated by the receiver operating characteristic (ROC) curve.Results The expression levels of LINC00978 in MGC-803 cells were significantly higher than that in GES-1 cells (18.88 ± 1.15 vs 1.00 ± 0.03,t =-21.926,P ＜ 0.05).The expression levels (median[P25,P75]) of serum LINC00978 in gastric cancer patients (3.525 [1.385,8.954]) were significantly higher than those in the patients with gastric benign diseases (0.419 [0.258,1.369],Z =-4.834,P ＜ 0.01) and healthy controls (0.814 [0.351,2.510],Z =-4.686,P ＜ 0.01).The expression levels of serum LINC00978 in gastric cancer patients were significantly related to lymphatic metastasis (r =0.448,P ＜ 0.01),invasive depth (r =0.369,P ＜ 0.01) and TNM stage (r =0.383,P ＜ 0.01),and those after operation significantly lower than before operation (0.17 [0.15,0.39] vs 0.98 [0.59,1.61],Z =-5.731,P ＜ 0.01).There was no significant difference in serum LINC00978 levels between the patients with gastric benign diseases and healthy controls (Z =-1.693,P ＞ 0.05).The area under the ROC curve (AUCROC) and 95％ confidence interval (CI) of serum LINC00978 levels in the diagnosis of gastric cancer were 0.807 and 0.732-0.882,respectively.When the cutoff value was 1.806,the sensitivity and specificity for the diagnosis of gastric cancer were 71.4％ and 75.9％,respectively.Conclusion The expression levels of serum LINC00978 in gastric cancer patients increase and are related to lymphatic metastasis,invasive depth and TNM stage,which may be served as a novel biomarker for the diagnosis of gastric cancer.

Objective To investigate the role of c-Jun N-terminal kinase (JNK1/2) signaling pathway in enterovims 71 (EV71) infection.Methods The effects of different concentrations of SP600125 on the activity of human rhabdosarcoma (RD) cells were detected by trypanbalu staining.The levels of VP1 mRNA and protein in EV71-infected RD cells were detected by real time Q-PCR and western blot,respectively.The levels of total and phosphorylated JNK1/2,c-Fos and c-Jun protein were determined by western blot.Last,the effects of.JNK1/2 inhibitor SP600125 on EV71 replication and JNK1/2 signaling pathway were analyzed.Results The results of trypanbalu staining showed that 5 and 10 μmol/L of SP600125 didn't influence on the activity of RD cells (P ＞ 0.05),while 20 μmol/L of SP600125 decreased the survival of RD cells significantly (P ＜ 0.05).Compared with the control,the expression levels of VP1 mRNA and protein in EV71-infected RD cells decreased obviously at 8 hours post-infection (P ＜0.01).In addition,after RD cells were infected EV71,the levels of phosphorylated JNK1/2,c-Fos and c-Jun increased significantly (P ＜ 0.05).However,the pretreatment of SP600125 decreased the phosphorylation levels of JNK1/2,c-Fos and c-Jun protein obviously (P ＜ 0.05).Conclusion EV71 infection may effectively activate the JNK1/2 signaling pathway in RD cells,which may be related to EV71 replication.

Objective To detect altered levels and clinical significance of serum miR-216a and prognosis monitoring in acute pancreatitis (AP) patients.Methods Serum miR-216a levels were determined by quantitative reverse-transcription PCR (qRT-PCR) assay among 80 mild acute pancreatitis (MAP) patients,80 severe acute pancreatitis (SAP) patients and 74 healthy controls.And amylase (AMY),lipase (LPS),Ca2+,glucose (Glu),hematocrit (HCT),triglyceride (TG),total cholesterol (TC) and C reactive protein (CRP) were measured by biochemical analyzer.The clinical usefulness of miR-216a for AP patients was assessed by ROC curve analysis and correlation analysis.Results Compared with healthy controls (11.12 × 10-5 [5.83 × 10-5,19.12 × 10-5],the serum miR216a levels were significantly increased in AP patients(38.49 × 10-5 [24.05 × 10-5,62.02 × 10-5],(U =-9.10,P ＜ 0.01) . The serum miR-216a levels in MAP and SAP patients were (36.46 × 10-5 [22.29 × 10-5,55.80 × 10-5] vs 40.44 × 10-5 [25.84 × 10-5,65.48 × 10-5]),there was no significant difference between MAP and SAP patients (U =-0.96,P ＞ 0.05).The areas under ROC curve (AUCROC) of miR-216a for differential healthy controls and AP patients was 0.870 (95％ CI:0.825-0.915),cut-off value is 0.61.AUCROC of miR-216a for differential healthy controls and MAP patients was 0.865 (95％ CI:0.808-0.921),cut-off value is 0.59.And AUCROC of miR-216a for differential healthy controls and SAP patients was 0.876 (95％ CI:0.822-0.930),cut-off value is 0.66.Moreover,after the clinical improvement of the patients,the levels of serum miR-216a were significantly lowered from (41.88 × 10-5 [24.24 × 10-s,64.44 × 10-5]) to (20.58 × 10-5 [11.01 × 10-5,41.91 × 10-5]),the differences was significant(U =5.24,P ＜ 0.0l).Correlation analysis showed that miR-216a was positively correlated with CRP (r =0.215,P =0.006) in AP patients.Conclusion The levels of miR-216a in serum of AP patients were increased,which is a potential biomarker for the diagnosis and prognosis monitoring of AP.