Objective This paper aims at establishing a inductively coupled plasma mass spectrometry ( ICP-MS) method for quantification and evaluation of iodine in human urine and serum in routine clinical laboratory .Methods This study was methodology validation research on iodine evaluation using ICP-MS.Ammonia, isopropanol and ultrapure water were mixed at certain ratio to dilute samples in the ratio of 1:10, and then the diluted samples were analyzed by ICP -MS.Re was used as the internal standard.And linearity, lower limit of detection, recovery, precision, accuracy, carryover and stability was evaluated thoroughly .Results of iodine of pregnant women who required iodine tests were retrospectively analyzed to evaluate the status of iodine .Results The method only needs 30s for analysis of one sample .It was sensitive with a lower limit detection of 0.87μg/L, the correlation coefficient was higher than 0.999 9 in ten measurements.The recovery in both serum and urine was approximately 100% (95.3% -109.9%). Based on the NIST standard reference material 3668 comparison, the bias was less than 4%( -0.9% -3.9%).The inter-coefficient variation (CV) for serum iodine and urine iodine was 1.2%-3.0%, 2. 0%-2.9%, respectively;and total CV for serum iodine and urine iodine were 3.0%-3.8%, 4.1%-4.9%, respectively.The mean carryover of this method was 0.03% and iodine was stable for at least one month at -20℃ and 4℃.The urine and serum iodine for pregnant women was (154.8 ±89.7) μg/L (mean ±SD),(75.8 ±21.4) μg/L, respectively.The correlation between urine and serum iodine was 0.21. Conclusion Establishe a rapid and simple ICP -MS method for urine and serum iodine measurement with high accurate and precise in routine clinical laboratory .

Objective To assess the interference by calcium dobesilatein 7 peroxidase-baseduric acid assays and to determine its clinical significance.Methods In the in vitro experiments, uric acid in pooled serum with final concentrations of calcium dobesilate additions (0, 2, 4, 8, 16, 32, and 64μg/ml) were measured by 7 peroxidase-based assays.Percent Bias (%) was calculated relative to the drug-free specimen.In the in vivo experiments, changes in serum uric acid and calcium dobesilate concentrations were observed before and after calcium dobesilate administration ( baseline, 0 h, 1 h, 2 h, 3 h, 4 h, 6 h ) involunteers.The interference in different assays was assessed compared with LC-IDMS/MS method. Calcium dobesilate levels in 40 specimens from those taking calcium dobesilate were measured by HPLC method.Of the 40 specimens, 10 were selected to analyse the levels of uric acid by both peroxidase and UV measurement method to assess the impact in clinical status.Results In the in vitro study, concentrations of uric acid measured by 7 peroxidase-based assays were reduced by -6.3%to -21.2%compared with drug-free serum, when theconcentration of calcium dobesilate was16μg/ml.In the in vivo study, comparedto UA levels at 0 h, the biasesof serum uric acid determined by peroxidase method after calcium dobesilate administration(1 h, 2 h, 3 h, 4 h, 6 h) were of -3.33%, -6.79%, -7.49%, -6.07%, -4.09%, respectively.The observed uric acid concentrations for 8 participants measured by enzymatic assays were inhibited by -3.75% to -6.89% at 0 hour and by -16.9% to-22.22% at 2 hours relative to the concentrations measured by the LC-IDMS/MS method. Conclusions Calcium dobesilate produced a clinically significant negative interference with uric acid in all peroxidase-based uric acid assays,which may result in false evaluation of uric acid level in clinical status.Significant differences in the degree of interference were observed among the assays.

OBJECTIVETo develop monoclonal antibodies against the catalytic subunit of human telomerase hTERT for its expression detection of human tumors.

METHODSA dominant epitope in hTERT (peptide hTERT(9))was automatically synthesized based on Fmoc method, and was used to immunize BALB/c mice. Hybridomas were generated and screened by ELISA for specific monoclonal antibodies, and the characterization of which were performed by Western blotting and immunohistochemical staining.

RESULTSAntigenic peptide hTERT(9) was synthesized and confirmed by MALDI-TOF-MS and HPLC analysis. Three hybridoma cell lines secreting anti-hTERT(9) antibodies designated as H4, G8 and A11 were established after primary screening and consequent three rounds of limited dilution. Both of H4 and G8 were IgM, while A11 was IgG1 in isotyping. The competitive assay showed that the antibodies were hTERT(9) specific, and the affinity of G8 was stronger than that of H4 and A11 assayed by affinity ranking. However, in Western blotting, both of H4 and G8 stained an about 123 000 protein band with HeLa and 293 cell extracts but not with normal 2BS cells. Besides, positive staining presented in the nucleus of HeLa, while 2BS was non-reactive immunohistochemically. The sections from paraffin-embedded blocks of 127 cases of human cancer, 40 of precancerous and 19 of benign tumors were in situ stained by G8 antibody, the results showed that the human cancer tissues were 80.31% (102/127) positive in specific nuclear reaction, on the contrary, only a minority of precancerous lesions present weak positive (17.5%, 7/40), and negative in benign tumors (0/19).

CONCLUSIONSThe monoclonal antibodies developed against synthetic peptide were hTERT-specific and could recognize both the native and the denatured form. Thus their use in immunoblotting or immunohistochemistry for detecting the telomerase hTERT expression of cancer cell and tissues was promising.

Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Binding, Competitive ; Blotting, Western ; methods ; Catalytic Domain ; DNA-Binding Proteins ; Female ; HeLa Cells ; Humans ; Immunohistochemistry ; methods ; Mice ; Mice, Inbred BALB C ; Neoplasms ; enzymology ; pathology ; Telomerase ; chemical synthesis ; immunology

Objective To validate the performance of six enzymatic glycated albumin reagents and evaluate their clinical application.Methods The performance of six enzymatic glycated albumin reagents(labled as A,B,C,D,E,F) from Beijing Jiuqiang Co, Beijing Lideman Co,Ningbomeikang Co, Beijing Haomai Co, Sichuan Maike Co and Asahi Kasei Co were assessed on Olympus AU5800 automatic biochemistry analyzer.According to the standard of CLSI,the precision,interference and linear correlation of these reagents were assessed.To assess the accuracy of GA% ,we used GA standard material whose value had been assigned using ID-LC/MS method provided by ReCCS.To do the method comparison and determine the consistency of assay, 50 fresh serum samples of T2DM outpatient and 80 fresh serum samples of apparently healthy people in Jan 2016 were tested using six kits.According to the EP28-A3C protocol, the reference range for GA%was validated in 122 apparently healthy individuals undertaking medical examination from January to February 2016 in PUMC.Results The precision,and the ability of anti-interference of the six reagents were good.The accuracy percentage deviation of six reagents was-19.3%-9.2%.The correlation coefficient of domestic reagents A to E and imported reagents F in the determination of GA% was 0.966-0.999, the average absolute bias was 7.0%-10.4%.The coincidence rate of A-E and F in determining abnormal GA% was between 88.5% and 96.9%.The coincidence rate was increased after switching to the reference range for preliminary clinical evaluation.Conclusion Six GA enzymatic kits used in automatic biochemical analyzer have high precision and strong anti-interference ability.Accuracy still needs to be improved.

A 72-year-old female patient was admitted to our hospital due to chronic cough, expectoration and dyspnea for 10 years,aggravated with intermittent fever for 1 month. She was diagnosed as acute exacerbation of chronic bronchitis,chronic pulmonary heart disease,cardiac function class IV,bilateral lower extremity venous thrombosis and thrombocytopenia. The application of antico-agulant drugs and coagulant drugs in the patient needed to be well weighed with the methods of the bleeding score system combined with clinical assessment of actual risk of bleeding. The interactions of drugs harmful to the patient should be considered and the prognosis of the patient should also be evaluated with careful clinical thought to reduce the patient's risk.

Gastric cancer (GC) is one of the most resistant malignancy to several treatment strategies. In recent years, the morbidity and mortality of GC has stayed high. Surgical resection remains the main treatment option for GC at present. Although chemotherapy, radiotherapy, immunotherapy, and traditional Chinese medicine are used as adjuvant therapies after surgery, the prognosis and five-year survival rate are still low. Currently, there is no effective method for early diagnosis of GC and thus most patients are diagnosed only when the advanced symptoms appear. Exosomes contain and transfer DNA, RNA, proteins, lipids, and other biological macromole-cules. Several studies have found that exosomes are involved in tumor processes including cell proliferation and metastasis. In particu-lar, the abundant biological macromolecules present in the exosomes reflect the progress of tumor development thereby enabling them as non-invasive diagnostic markers. This provides a new idea for diagnosing GC in early stages. In this review, the contribution of exosomes to GC metastasis and the applications of exosomes in early GC diagnosis are briefly summarized.

Chimeric antigen receptor T (CAR-T) cell therapy is an emerging immunotherapy that has allowed for major breakthroughs in the treatment of hematological neoplasms. However, little progress has been made in the treatment of solid tumors, primarily due to the difficulty in homing to tumor tissues by CAR-T cells during treatment. The complex tumor microenvironment and the barrier function of tumor tissues prevent CAR-T cells from contacting tumor cells, thereby preventing them from exerting their antitumor ac-tivity. This review article summarizes not only the progress made in the study of homing disorders of CAR-T cells in the treatment of solid tumors but also the current methods to overcome these disorders.

A simple and rapid immunochromatographic test strip incorporating a colloidal gold-labeled recombinant Nsp7 antigen probe was successfully developed for the detection of anti-porcine reproductive and respiratory syndrome virus (PRRSV) antibodies in swine. Recombinant Nsp7 protein of PRRSV labeled with colloidal gold was dispensed on a conjugate pad for use as the detector. Staphylococcal protein A and purified porcine anti-Nsp7 antibodies were blotted on a nitrocellulose membrane to form test and control lines, respectively. A comparison of the strip with standard diagnostic tests, enzyme-linked immunosorbent assays and immunoperoxidase monolayer assay, was also performed. The immunochromatographic test strip was shown to be of high specificity and sensitivity. Furthermore, the strip assay is rapid and easy to perform with no requirement for professional-level skills or equipment. It is suggested that the immunochromatographic test strip can be used to quickly and accurately detect PRRSV antibody and to be suitable for diagnostic purposes in the field.
Antibodies* ; Collodion ; Colloids ; Diagnostic Tests, Routine ; Enzyme-Linked Immunosorbent Assay ; Gold Colloid ; Immunochromatography ; Membranes ; Porcine Reproductive and Respiratory Syndrome* ; Porcine respiratory and reproductive syndrome virus* ; Sensitivity and Specificity ; Staphylococcal Protein A ; Swine

Objective To investigate the effects and mechanisms of shRNA interfered with expression of leucine-rich repeat containing G-protein coupled receptor 5 (Lgr5) on the malignant behaviors of colorectal cancer stem cells (CSCs).Methods The experimental study was conducted.The CSCs expressing Lgr5+ were sorted by fluorescence activated cell sorting.Lgr5+ cells that were transfected with Lgr5-shRNA lentiviral vector and nontarget shRNA lentiviral vector were respectively allocated into the experimental group and control group.The percentage of Lgr5+ cells was analyzed by flow cytometery.The relative expression of Lgr5 mRNA was detected by fluorescence quantitative real-time polymerase chain reaction (qRT-PCR).The capacity of self-renewal was detected by sphere forming assay.The tumorigenesis in vitro and in vivo were respectively measured by colony formation assay and xenografting experiment.The mRNA expressions of stem cells related genes (Oct4,Sox2,Nanog,KLF4),CSCs genes (CD133,CD44,ALDH) and Wnt/β-catenin pathway key genes (Axin2,Wnt5a,Wnt3a,Fzd3,c-myc,VEGF,Ascl2,claudin-1) were detected by qRT-PCR.Measurement data with normal distribution were represented as-x±s.Comparison between groups was analyzed using the t test.Results (1)Transfection efficiency of shRNA lentiviral vector induced Lgr5 by flow cytometery was respectively 6.8％± 1.0％ in the experimental group and 92.7％±3.3％ in the control group,with a statistically significant difference (t =43.148,P＜0.05).The relative expression of Lgr5 mRNA measured by qPT-PCR was respectively 0.168±0.057 in the experimental group and 1.148±0.004 in the control group,with a statistically significant difference (t=28.778,P＜0.05).(2) The capacity of self-renewal was detected by sphere forming assay.The results of sphere forming assay:the number of spheres was 29±6 in the experimental group and 410± 10 in the control group,with a statistically significant difference (t =41.070,P＜0.05).The results of colony formation assay:the numbers of colonies in the experimental group and control group were respectively 72±4 and 412± 19,showing a statistically significant difference (t =31.433,P＜ 0.05).The results of tumorigenesis:the volumes of tumors in the experimental group and control group were respectively (81± 15)mm3 and (328±24)mm3,with a statistically significant difference (t=11.304,P＜0.05).(3) The effects of Lgr5 down-regulation on related genes,results of qRT-PCR detection:① The mRNA relative expressions of Oct4,Sox2,Nanog and KLF4 (stem cells related genes) were 0.377±0.093,0.662±0.104,3.591±0.300,0.425±0.091 in the experimental group and 1.957± 0.026,2.137±0.015,5.831±0.165,1.536±0.014 in the control group,with statistically significant differences (t=23.079,22.261,8.446,19.186,P＜0.05).② The mRNA relative expressions of CD133,CD44 and ALDH (CSCs genes) were 1.490±0.155,5.535±0.487,1.640±0.039 in the experimental group and 2.488± 0.061,9.908±0.332,5.718±0.292 in the control group,with statistically significant differences (t =8.170,9.667,27.849,P＜0.05).③The mRNA relative expressions of Axin2,Wnt5a,Wnt3a,Fzd3,c-myc,VEGF,Ascl2 and claudin-1 genes in the Wnt/β-catenin pathway were respectively 1.592±0.267,0.528±0.138,2.153±0.078,1.480±0.064,0.248±0.128,1.492±0.025,0.658±0.095,1.647±0.087 in the experimental group and 3.651±0.224,2.570±0.093,2.301±0.157,1.636±0.058,1.415±0.080,2.610±0.159,2.480±0.123,3.432±0.273 in the control group.There were statistically significant differences in the mRNA relative expressions of Axin2,Wnt5a,c-myc,VEGF,Ascl2 and claudin-1 genes between the 2 groups (t =7.316,15.332,12.649,12.320,14.831,9.063,P＜0.05),and no statistically significant difference in the mRNA relative expressions of Wnt3a and Fzd3 between the 2 groups (t =2.887,2.242,P＞0.05).Conclusion The malignant behaviors of colorectal CSCs are suppressed after shRNA lentivirus interfered with expression of Lrg5,and its mechanism is related to inhibiting activity of Wnt/β-catenin pathway.

Objective To investigate the changes of prevalence of hyperuricemia ( HUA) and its correlations with blood glucose and lipid in healthy adults receiving physical examination at Peking Union Medical College Hospital (PUMCH) from 2012 to 2017. Meth-ods An observational approach was adopted for the data analysis.The test results of uric acid (UA),fasting blood glucose (FBG),to-tal cholesterol (TC),triacylglycerol (TG),low density lipoprotein cholesterol (LDL-C),high density lipoprotein cholesterol (HDL-C), creatinine (Cr) and Urea of 399 089 cases (206 881 males and 192 208 females) at PUMCH from January 2012 to December 2017 were collected and statistically analyzed.Results The total prevalence of HUA was 17.4% in which the prevalence of males was signif-icantly higher than that of females (25.6% vs 8.5%,χ2＝20 234.850,P＜0.01).During the years of 2012 to 2017,the prevalence of HUA was 26.5%,24.7%,28.6%,23.9%,24.8% and 24.5% in males,and 13.8%,6.3%,7.9%,6.1%,6.2% and 6.8% in females for each year respectively.The prevalence of HUA in males aged 18 to 64 years old was significantly higher than that in the age-matched fe-males (all P＜0.05).However, the prevalence of HUA in males aged≥65 years old was similar to the age-matched females.There was no statistically significant difference of HUA prevalence between males and females aged ≥65 in 2013,2015,2016 and 2017 ( χ2＝1.792,0.017,1.440 and 0.205 respectively;all P＞0.05).The percentages of hyperlipidemia in both males and females of HUA group were higher than those of non-HUA group respectively (all P＜0.01).The percentage of hyperglycemia in males of non-HUA group was higher than that of HUA group,but the percentage of hyperglycemia in females of non-HUA group was lower than that of HUA group ( all P＜0.01).High levels of TC,TG and FBG were risk factors of HUA with increased OR values in increased concentrations of TC,TG and FBG,respectively.Conclusion During the recent 6 years, in healthy adults receiving physiced examination at PVMCH, the preva-lence of male HUA diagnosed was at overall high level,but the prevalence of female HUA was in decreasing and relatively stable trend. Hyperlipidemia and hyperglycemia should be the risk factors of HUA.