Objective To evaluate the performance of sST 2 ELISA kit and investigate the clinical application of sST2.Methods This verification study validated the precision , linearity of sST2 ELISA kit according to the CLSI EP-15A, EP-6A protocols.300 healthy adults(aged from 20 to 85, 124 male and 176 female) from 5 different districts of Shanghai were used to establish serum sST 2 reference interval .The correlations between sST2, NT-ProBNP, LVEF and NYHA class were analyzed in 117 patients diagnosed with heart failure who were grouped according to the New York heart association ( NYHA).Receiver operating characteristic (ROC) curve was used to compare the ablity of sST2, NT-ProBNP, LVEF in distinguishing heart failure patients .Results The within-lot and between-lot variation of three level samples were below 4% and 10% respectively.There was a good linear correlation ( Y=0.995X+0.005, R2 =0.999) between theoretical value and actual detection result in the range of 0 to 200 μg/ml.The reference interval of sST2 was 10.2 to 41.0μg/ml for males and 8.9 to 28.1μg/ml for females.sST2 was positively correlated with NT-ProBNP and NYHA class but did not correlate with LVEF in heart failure patients . Patients with NYHA class>II (Median:28.3,IQR:19.5-39.2)had higher serum sST2 level than patients with NYHA class≤II (Median:45.1,IQR:34.1 -85.6), P<0.05.The AUC of sST2 in distinguishing heart failure patients from normal people was 0.815(sensitivity :51.2%,specificity:92.7%).The AUC of sST2 ,sST2+NT-ProBNP and sST2+NT-ProBNP+LVEF in distinguishing patients between NYHA class≤II and>II were 0.743, 0.810, 0.831 respectively and the sensitivity of sST 2 +NT-ProBNP+LVEF was 94.7%.Conclusions Experimental results show that this sST 2 ELISA kit has a good performance in the precision, linearity.sST2 correlates with NT-ProBNP and NYHA class but do not correlates with LVEF . Serum sST2 level is not influenced by age , BMI, renal function.sST2 could be a good supplement of NT-ProBNP and LVEF in distinguishing patients between NYHA class≤II and>II.

Objective To evaluate the performance of serum sialic acid detection kit using enzymic method and investigate the clinical diagnosis value of sialic acid.Methods one hundred and fifty healthy adults were enrolled in this case control study to establish serum SA reference interval.The analytical performance (accuracy,precision,linearity) of serum sialic acid detection kit using enzymic method was assessed.Two hundred and forty patients were classified into different malignant tumor groups according to their pathological types.Serum SA level of each tumor group was compared with that of normal control group.In tumor groups with statistical difference,benign disease groups were further collected.Receiver operating characteristic (ROC) curve and area under curve (AUC) were used to evaluate the diagnostic value of SA compared with other tumor markers.t test,one-way ANOVA,Mann-Whitney U test were used as statistical methods.Results The reference interval of SA was 479 to 715 mg/L.The detection result of 2 level controls was 584 and 1 482 mg/L respectively,which were both within the acceptable limits.The within-lot and between-lot variations of three level samples were both below 5％.There was a good linear correlation (Y =0.995X-0.177,R2 =0.999) between theoretical value and actual detection result in range of 0-1 052 mg/L.The serum level of SA was (757 ± 177),(514 ± 86) and (597 ± 60) mg/L in gastric cancer group,benign disease control group and normal control group respectively,which had statistically significant difference(F =55.2,P ＜ 0.01).The serum level of SA was(659 ± 127) and (545 ± 66) mg/L in colorectal cancer group and benign disease control group respectively,which had statistically significant difference(F =42.8,P ＜ 0.01).The serum level of SA was (738 ± 157) and (672 ± 161) mg/L in colorectal cancer group and benign disease control group respectively,which did not have statistically significant difference(F =26.3,P ＞ 0.05).The AUC of SA was 0.804,0.724,0.755 in gastric cancer group,colorectal cancer group and lung cancer group respectively,which was higher than that of CEA and CA72-4.In gastric cancer group,the sensitivity of SA was higher than that of CEA (59.5％,24.3％).The AUC of SA was 0.791,0.687,0.790 in gastric cancer,colorectal cancer and lung cancer patients with normal CEA serum level respectively.Conclusions Experimental results show that serum sialic acid detection kit using enzymic method has good performance in the precision and linearity.Sialic acid has some value in the diagnosis of gastric cancer and colorectal cancer and could be a good supplement of CEA in screening of cancer.

Objective To investigate the relationship between corneal basal nerve change and type 2 diabetic retinopathy based on confocal laser microscopy.Methods Together 118 patients with type 2 diabetes (T2D) were collected in our hospital from February 2016 to February 2017,including 57 patients with diabetic retinopathy (DR group) and 61 patients without DR (NDR group).For comparison,60 healthy volunteers were selected as the control group.And all the subjects were examined by corneal confocal laser microscopy to analyze the relationship between the morphological parameters of the corneal nerve and clinical variables.Results Corneal nerve fiber density,corneal nerve branch density and corneal nerve branch length in DR group were (20.03 ±4.22) · mm-2,(22.01 ± 7.05) · mm-2 and (9.50 ± 1.76) mm ·mm-2,significantly less than those of the control group and NDR group (all P ＜ 0.05);and corneal nerve fiber curvature was (0.30 ± 0.03),significantly higher than that of the control group and NDR group (all P ＜ 0.05);In DR patients,phase Ⅲ patients had smaller the corneal nerve fiber density,corneal nerve branch density and corneal nerve branch length,but the larger corneal nerve fiber curvature than the phase Ⅰ and Ⅱ patients (all P ＜ 0.05);course of disease of DR group was (12.04 ± 2.48) years,which was significantly higher than that of NDR group (P ＜ 0.05),while fasting C peptide and fasting insulin were (1.41 ± 0.58) μg · L-1 and (20.05 ± 7.91) mU · L-1,respectively,significantly lower than those of NDR group (all P ＜ 0.05);The duration of T2D was negatively correlated with the corneal nerve branch density and corneal nerve branch length (r =-0.322,-0.317,all P ＜0.05);Fasting C peptide was positively correlated with the corneal nerve branch density (r =0.298,P ＜ 0.05),and negatively correlated with the corneal nerve curvature (r =-0.311,P ＜ 0.05).Conclusion Patients with T2D retinopathy have abnormal morphology of corneal nerve.And confocal laser scanning microscopy is conducive to the early detection of microvascular disease in T2D patients with a longer course of disease or a low level of fasting C peptide.

Objective:To improve previous method of primary rat cortical neuron culture to get purer and more long-lasting cells for study.Methods:Timed-pregnant Wistar rats at a gestational age of 16 or 17 days(16-17 d) were used.Fetal brains were removed and the cerebral cortices were dissected out.Papain digestion and mechanical dissociation were combined to conduct mono-cell suspending media.Four to six hours(4-6 h) post-plating,all plating media were removed from cultures and replaced with Neurobasal medium supplemented with B27.On the third day,10 ?mol/L cytosine arabinoside(Ara-C) was added to the culture for 24 h to inhibit the outgrowth of glial cells.Half of the culture medium was changed every week.The morphological changes of neuron cells were observed by light microscope.Double immuno-staining of microtubule-associated protein 2(MAP2) and karyon were applied to assess the culture purity.Evaluation of synapse formation was processed by immunocytochemical analysis using antibodies against both pre-and postsynaptic protein markers.Results:The improved method could remarkably increase the cell number and reduce neuronal damnification.The primary culture was characte-rized by high uniformity,purity,normal synapse formation and longtime livability.Conclusion:This is a simple and reliable technique for the in vitro primary culture of rat cortical neurons.

Objective:To find the discrepancies caused by HLA B gene variants in HLA B antigen homozygotes which were typed again by DNA method.Methods:75 blood samples assigned HLA B homozygotes by serology were typed and detected HLA B expression variants by PCR SSP.Results:The 12 samples had been assigned the second alleles by DNA method in 75 HLA B homozygotes by serology.One out of 12 samples was identified expression variant by PCR SSP.The null allele was caused by the insertion of an extra cytosine at the beginning of exon 4.Conclusion:HLA B gene typing by PCR SSP will be the use of supplement to serology.The expression variant detection by PCR SSP will improve accuracy for tissue typing and benefit clinical application.

Objective To develop an accurate,rapid,high throughout genotyping method based on oligonucleotide microarray for cytochrome P450 gene polymorphisms related to paclitaxel metabolism.Methods The mutant points of 2C8 * 3,3A4 * 18 and 3A5 * 3C from cytochrome P450 gene were regarded as targets.Based on the sequences in the GenBank,the wild-type and mutant-type probes were specially designed for each mutant point.PCR primers were located in the both sides of mutant point,and furthermore the fragments of PCR products were less than 200 bp.Each type of standard plasmids was constructed.Thus,all the olignucleotide probes were modified with 3'amino-group,and the reverse primers were labeled with fluorescein (Cy3).The probes were immobilized onto certain glass slides.The specific fragments of three genes were amplified and then hybridized with oligonucleotide microarray.The results were analyzed by using certain software.Finally this assay was applied to detect 50 clinical blood specimens.Results When PCR products from standard plasmids were hybridized with DNA microarray,the corresponding probes produced positive signals.Meanwhile,the non-specific hybridization signals did not appear.The results of clinical specimens showed that the mutant rate of CYP2C8 * 3 was 2%.The point of CYP3A4 * 18 for all the clinical specimens was wild-type and the mutant rate of CYP3A5 * 3C was 62%. Meanwhile,the results from detecting 50 clinical blood specimens using oligonucleotide microarray were the same as sequencing analysis.Conclusions Oligonucleotide microarray is a reliable and accurate genotyping assay for cytochrome P450 2C8 * 3.3A4 * 18 and 3A5 * 3C polymorphisms related to paclitaxel simultaneously.This genotyping assay is a high-throughout method for guiding personalized therapy and analyzing metabolism of paclitaxel in vivo.

Objective To evaluate the short-term prediction of high-sensitivity cardiac troponin T (hs-cTnT) and other cardiovascular risk biomarkers in patients undergoing maintenance hemodialysis (MHD).Methods We conducted a cohort survey in 296 consecutive MHD patients whose clinical data were retrospectively analyzed.Before MHD,hs-cTnT and other relative cardiovascular biomarkers were detected.The end point (all-cause death) and time of occurring were recorded in the next 13 months.The differences between survival and all-cause death were analyzed by t-test,Mann-Whitney test and x2 test.The best two percentile cutoff point was calculated by X-tile and the survival rate was calculated by Kaplan-Meier Logistic regression analysis was applied to analyze the odd ratio between high risk and non-high risk hs-cTnT group.Non-high risk group was divided into intermediate risk and low risk group based on the 99th percentile of hs-cTnT in healthy population,to further evaluate its short-term prediction value for MHD patients.The short-term significance of hs-cTnT was proved to be independently associated with all-cause death by Logistic regression analysis.Results The mean value of serum hs-cTnT in survival group was 0.05 (0.03～0.07) ng/mL,while in the death group it was 0.07 (0.04～0.14) ng/mL,which had statistical significance (P =0.027).The best two percentile cutoff of hs-cTnT in MHD patients was 0.1 ng/mL.The survival rate in high risk group (hs-cTnT＞0.1 ng/mL) is lower than it in non-high risk group (hs-cTnT≤0.1 ng/mL) (76.67％ vs.96.62％,P ＜0.05).The odd ratios for high risk group and non-high risk group was 7.288 (P＜ 0.001).Moreover,further grouping the non-high risk group by hs-cTnT =0.014 ng/mL,intermediate risk group (hs-cTnT＞0.014 ng/mL) group has lower survival rate than low risk group (hs-cTnT≤0.014ng/mL),while there wasn't any death case occurred in the low risk group.Conclusions Hs-cTnT is an independent risk factor to all-cause death.Thus hs-cTnT can be a strong indicator of short-term prediction and prognostic evaluation.

Objective To evaluate the performance of serum small dense low-density lipoprotein cholesterol(sdLDL-C)kit using enzymic method and evaluate the relationship with the severity of coronary heart disease.Methods Performance verification methodology. The analytical performance consisted of accuracy, precision and linearity of serum sdLDL-C kit using enzymic method was assessed. One hundred and twenty healthy persons were recruited to establish serum sdLDL-C reference interval. Two hundred and twelve patients underwent coronary angiography were enrolled in the study.Among them 110 cases were positive for coronary angiography, where as 102 cases were negative. We examined serum levels of sdLDL-C in 110 patients with positive angiography, 102 patients with negative angiography and 120 healthy volunteers. Positive group was classfied into severe group(Gensini score>30) and mild group (Gensini score≤30).Results The accuracy and precision of sdLDL-C examination were in compliance with manufacturer′s statement and there was a good linear correlation(Y=0.9937X-0.1063,R2=0.99) in range of 0.06-2.45 mmol/L. The reference interval of sdLDL-C was 0.15-0.97 mmol/L and without gender and age specificity. The level of sdLDL-C was higher in positive angiography group than in negative angiography group and healthy control group(P<0.01). The level of sdLDL-C was higher in severe group than in mild group(P<0.05). Binary stepwise regression analysis demonstrated that sdLDL-C was independently associated with the severity of coronary heart disease(OR=3.101,P<0.05).ConclusionsExperiment data demonstrated that serum sdLDL-C kit using enzymic method has good performance in the accuracy, precision and linearity. SdLDL-C that plays an important role in the occurrence and progression of coronary heart disease, is an independent important risk of the severity of coronary heart disease.

Objective To investigate the clinical and laboratory profiles of a patient with pustular psoriasis-like skin lesion and cerebral palsy due to biotinidase deficiency. Methods A 5 year and 4 month-old boy with biotinidase deficiency was confirmed by urinary organic acid analysis with gas chromatography/mass spectrometry (GC/MS)and biotinidase activity assay of peripheral blood. His clinical features, laboratory findings, treatment and outcome were studied. Results The boy showed difficulty in taking food after birth, gradually eczema and pustules appeared at the age of 2 months, and generalized erythema and intractable pustular psoriasis-like lesion at the age of 8 months. His intellectual development was normal with retardation of locomotor system. He had muscular dystonia at the age of 6 months. Physical examination showed generalized pustular psoriasis-like lesion, generalized paralysis, hypertonic contracture of extremities, sparseness of scalp hair and severe malnutrition. Routine laboratory tests showed a mild anemia, metabolic acidosis and elevation of plasma creatine phosphokinase. Increased excretion of urinary lactate, pyruvate, 3-OH-propionate, propionylglycine, and 3-methylcrontonylglycine were observed. Biotinidase activity of his peripheral blood was below 0.1 pmol/min/3mm (normal 6.3-9.3 pmol/min/3mm). Biotin (10 mg/day) supplementation led to a dramatic recovery of the skin lesion. After the treatment of rehabilitation, his muscle power was also improved gradually. Conclusions Dermatological and neurological manifestations are the main features of biotinidase deficiency. Early diagnosis and biotin administration can greatly improve the clinical symptoms. Generalized pustular psoriasis-like lesion and cerebral palsy of this boy have improved after the supplementation of biotin, but he may be remained wheelchair-dependent because of delayed diagnosis.

Objective Digital PCR ( dPCR ) was established to detect plasma epidermal growth factor receptor (EGFR) T790M mutation of non-small cell lung cancer (NSCLC) patients and was evaluated in terms of analytical performance and clinical application significance.Methods The specific primers and probes for EGFR T790M mutation and wildtype were designed to establish dPCR platform.Limit of blank, sensitivity and linearity of dPCR were evaluated by the detection of plasmids with different concentrations to set up optimal reporting system and reanalyzing process.The mutation of EGFR T790M in plasma and tissue samples from 10 patients with advanced NSCLC resistant to EGFR-TKI therapy who were enrolled in Zhongshan Hospital Fudan University from January 2014 to October 2015 were analyzed by dPCR and amplification refractory ( ARMS) , respectively.The consistency was evaluated between dPCR and ARMS by Chi-square test.The correlation of T790M abundance detected by dPCR between plasma and tissue samples was also analyzed by Peasrson correlation analysis.Results Limit of blank and sensitivity of dPCR was 10 copies and 0.01%, respectively.dPCR was evaluated as linear in the range of 0.01%-100%( Y=1.226X-3.984,R2 =0.999 ).The consistency between dPCR and ARMS of tissue samples was good ( kappa=0.80), while the positive rates of plasma T790M detected by dPCR was significantly higher than ARMS (50%vs 20%,P<0.05).It was found that T790M abundance detected by dPCR was highly correlated between lung cancer tissue and plasma ( R =0.923, P <0.05 ) using Pearson correlation analysis. Conclusions A new method of dPCR with high sensitivity and absolute quantification is established for the detection of EGFR T790M mutation in plasma from advanced NSCLC patients, which brings tumor liquid biopsy into real.It has the ability to provide the most direct and valuable guidance for clinicians to make decision on EGFR tyrosine kinase inhibitors therapy in patients with advanced NSCLC resistant to EGFR-TKI.