1.Establishment of a digital PCR platform for detection of EGFR T790M mutation in plasma circulating DNA from NSCLC patients
Fei HUANG ; Qian YU ; Jiong WU ; Shengchao WU ; Beili WANG ; Chunyan ZHANG ; Wei GUO ; Paishen PAN
Chinese Journal of Laboratory Medicine 2016;39(3):170-175
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.
2.Establishing reference intervals of the routine laboratory tests for hepatorenal function in Shanghai by biochemistry
Wenhao WU ; Junfei SHEN ; Jiong WU ; Chunyan ZHANG ; Beili WANG ; Wei GUO ; Baishen PAN
Chinese Journal of Laboratory Medicine 2016;39(12):906-910
Objective To establish reference intervals of nine laboratory projects for hepatorenal function and verify its reliability by indirect method . Methods ALL test results were extracted from physical examinations that were stored in the laboratory information system of Zhongshan Hospital during 2012 to 2014.Using Skewness-Kurtosis test to detect the normality of data , if not the original data were transformed through BOX-COX transformation to obtain an approximatenormal distribution .Outliers were identified and omitted by Turkey method .The indirect reference intervals were taken by applying Hoffmann method.The reference change value ( RCV) was selected to inspect the statistical significance between the calculated and published reference intervals .Results Among those nine laboratory projects ,thedifferences betweenthereference intervalsfor ALT , AST, AKP, GGT, TP, BUNwerelessthan their RCV ( <60.12%, 38.12%, 19.9%, 41.53%, 8.53%, 37.5%) , there were no significant differences between the direct method.There were slight differences between the calculated reference intervals and published reference intervals for the lower limit of the LDH and the upper limit of the ALB (>26.65%,9.92%) ,and there was a significant difference between the calculated reference intervals and the current reference interval in the laboratory for the lower limit of the UA of male (>26.65%).Conclusion This research further proved the reliability of indirect reference intervals and this technique deserved to be promoted and applied by other clinical laboratories.
3.Clinical Application Value of the Result of the HCV RNA,FIB-4 and Levels of 2 5-Hydroxy Vitamin D for Patients with Hepatitis C
Yan ZHOU ; Wenhao WU ; Jiong WU ; Chunyan ZHANG ; Beili WANG ; Wei GUO ; Baishen PAN
Journal of Modern Laboratory Medicine 2017;32(1):30-33,37
Objective To analyze the effects of 25-hydroxyvitamin D[25(OH)D]on the result of the HCV RNA and the FIB-4 in the patients with hepatitis C.Methods 255 serum samples were random collected from the patients with hepatitis C and 218 serum samples were random collected from the healthy people.The 25(OH)D,HCV RNA,aspartate aminotransferase (AST),alanine aminotransferase (ALT)and blood platelet (PLT)were detected.Then,compared the results of the 25 (OH)D in the patients with hepatitis C and the healthy group.Analyzed the relevance between the concentration of 25(OH) D and HCV RNA.According to the quartile concentration of the 25(OH)D,the patients with hepatitis C were categorized to four groups.The relationship of FIB-4 between HCVRNA and 25(OH)D was analyzed.Results The average concentration of the 25(OH)D in the patients with hepatitis C and healthy people were 48.16±1.41 nmol/L vs 60.42±1.34 nmol/L, with a significant difference (t=4.682,P<0.01).There were 38 patients (14.90%)had severe deficiency of 25(OH)D (<25 nmol/L)in 255 patients with hepatitis C.And there were 8 patients (3.67%)had severe deficiency of 25(OH)D (<25 nmol/L)in 218 healthy people,with a significant difference (t=5.216,P<0.01).Then found no relevance between the log-arithmic of the HCV RNA and the concentration of the 25(OH)D (r2=0.018 8,P=0.412)and there was significant differ-ence between the proportion of FIB-4 in the highest quartile concentration of the 25(OH)D and the lowest quartile concen-tration of the 25(OH)D (χ2=8.190,P=0.042).Conclusion The patients with hepatitis C were easier to have a severe de-ficiency of 25(OH)D than the healthy people.The hepatitis C patients should been suggested to supply the vitamin D.FIB-4 has a significant difference with 25(OH)D and no great effects on the result of the HCV RNA.
4.Molecular cloning of farnesyl pyrophosphate synthase from Alisma orientale (Sam.) Juzep. and its distribution pattern and bioinformatics analysis.
Wei GU ; Qinan WU ; Jianguo CHAO ; Beili XI ; Lin LI ; Xiuyuan SHEN
Acta Pharmaceutica Sinica 2011;46(5):605-12
Triterpenes, which have large application potential in the treatment of cancer, are the main active components of genuine medicinal material Alisma orientale (Sam.) Juzep. Farnesyl pyrophosphate synthase (FPPS) is one of the important rate-limiting enzymes in the synthetic pathway of triterpenes. In this study the FPPS full length cDNA of the A. orientale, was cloned via homology-based cloning approach and rapid amplification of cDNA ends (RACE). The full length of the FPPS cDNA was 1 531 bp (accession no. HQ724508), which contained a full 1 032 bp ORF that encoded 343 amino acids. The deduced protein sequence exhibited five conserved motifs, two of which is riched of Asp (DDXXD). The result of real-time quantitative PCR (QRT-PCR) showed that FPPS gene was expressed in different organs of A. orientale. The expression increased from October to the first ten-day period of December, and then decreased. The FPPS gene expression was higher in leaves but lower in leafstalk, tuber and root. HPLC analysis of active components 23-acetyl-alismol B of A. orientale. during different periods indicated that its change trend should be consistent with FPPS gene expression. It can be primarily deduced that FPPS gene should be an important control point in the synthetic pathway of Alisma terpenes. This study may facilitate the quality of medicinal plants through gene engineering in the future.
5.Harmonization of serum protein measurements with different immunoassays
Lin SUN ; Wei GUO ; Chunyan ZHANG ; Jiong WU ; Binbin SONG ; Beili WANG ; Baishen PAN
Chinese Journal of Laboratory Medicine 2013;36(9):839-844
Objective To analyze differences of serum proteins measurements by different methods in different systems and investigate whether the assigned serum valued by ERM-DA470k/IFCC can replace ERM-DA470k/IFCC in clinical measurements to promote the harmonization of serum proteins measurements.Methods Serum specimen was collected from 100 medical examination persons in Zhongshan Hospital (Jun 2012; age:27-73; 47 male and 53 female).Serum IgA,IgG,IgM,C3c,C4,TRF and PA of health persons,ERM-DA470k/IFCC and the assigned valued by ERM-DA470k/IFCC were measured by Hitachi 7600 (Roche and DiaSys reagents),Siemens BN Ⅱ (Siemens reagent) and Beckman IMMAGE (Beckman reagent),respectively.ERM-DA470k/IFCC was used as a calibrator to value the mixed serum.Serum proteins results obtained from the same methods were compared by T test and Wilcoxon test,and analysis of variance and Firendman test were used to compare results from different methods.The correlation of two groups was analyzed by linear regression.Results The differences of serum proteins results measured by two immunonephelometries,two immunoturbidimetries and all assays were statistically significant (P <0.05),respectively.The serum proteins results measured by two immunoturbidimetries showed good correlation (R2 were all above 0.97 except for C3c).In contrast,the serum proteins results measured by two immunonephelometries show poor correlation (R2 were all ranged from 0.90 to 0.94) except for IgM (above 0.96).The serum IgA,IgG and IgM results measured by different methods show good correlation (R2 were all above 0.95) Based on certified values of ERM-DA470k/IFCC,serum protein results measured by different methods could be harmonized,and got similar results from the assigned serum (P > 0.05).Conclusions There is significant difference of serum proteins results detected by different methods.The ERM-DA470k/IFCC and certified serum can make serum protein results obtained from different methods harmonized.
6.Predication values of white blood cell following chemotherapy in older patients with acutemyeloid leukemia
Xiaotao WANG ; Beili CHEN ; Wenyuan LIN ; Feng LIU ; Hong WU ; Donghua MO
Clinical Medicine of China 2009;25(6):573-576
Objective To explore the predictive value of Kinetics of white blood cell (WBC) elimination following induction chemotherapy for eider with acute myeloid leukemia(AML). Methods 71 elder with AML were reviewed. Chi-square and the Kaplan-Meier methods were used to identify the relationship between the nadir WBC count and time to WBC nadir with efficacy and survival. Results 28 patients (39.44%) achieved a complete re-mission(CR),another 19 (26.76%) had a partial remission,17(23.94%) patients had a non-remission,and 7 pa-tients(9.86%)died. Overall survival over 3 years was about 11.27% (8 cases),over 2 years about 23.94 % (17 cases),and over 1 years about 47.89 % (34 cases). The low WBC nadir and high WBC nadir in the bone marrow CR (9.86% and 12.68%)and the total survival rate (8.45% and 11.27%)were lower than the median absolute WBC nadir (16.90%, 15.49%), but were no statistical discrepancy (χ23.32,1.22, P=0.77, 0.54). The pa-tients who achieved WBC nadir in less than or equal to 10 days in the bone marrow CR(12.68%) and the total sur-vival rate(8.45%) were statistical significantly higher than those achieved it in greater than 10 days(26.76%). The patients whose WBC attained at low level less than or equal to 3 days in the bone marrow CR(16.90%) and the total survival rate(12.68%) were statistical significantly higher than those greater than 3 days (22.54%) (χ2 15, 57,11.71,4.85,9.54,P=0.001,0.01,0.03,0.04). Conclusion WBC nadir in loss than or equal to 10 days and WBC attained at low level greater than 3 days may serve as a worse prognosis.
7.The analytical performance assessment and clinical practice of three sensitive cardiac troponin Ⅰ assays
Chunyan ZHANG ; Lingyan SONG ; Jiong WU ; Binbin SONG ; Beili WANG ; Bin TANG ; Wei GUO ; Baishen PAN
Chinese Journal of Laboratory Medicine 2010;33(9):819-824
Objective To assess the analytical performance of three sensitive cardiac troponin Ⅰ assays and compare the clinical application to provide help in choosing the detection method for clinical laboratory. Methods A total of 474 serum samples were collected from apparently healthy subjects and a total of 112 serum samples were collected from patients presenting with symptoms suggestive of acute myocardial infarction. The functional sensitivities of three assays from Abbott, Beckman-Coulter and Ortho were determined ( CV = 10% ). The reference ranges have been established. The analytic performance was compared according to the assessment mode described by Apple. The relationship was compared among the different assays. The preliminary clinical application value for different detection methods has been evaluated and validated with self-established reference ranges. Results The functional sensitivities ( CV = 10% ) of the cTnI assays for Abbott, Beckman-Coulter and Ortho were 0. 030, 0. 04 and 0. 013 μg/L, respectively.The 99th percentiles of cTnI in healthy volunteers were 0. 021, 0. 02 and 0. 026 μg/L respectively. The analytical data of ROC curve showed that the area under curve (AUC) of the cTnI assays for Abbott,Beckman-Coulter and Ortho for diagnosis of AMI was 0. 852,0. 909 and 0. 910,respectively. There was no statistical difference between any two methods(Z1 = 1.18 ,Z2 = 1.21 ,Z3 =0. 026,all P >0. 05). There were good consistency between the 99th percentile obtained from our laboratory and suggested by manufacturers (Kappa value were 1. 000, 0. 730 and 0. 893 respectively, all P < 0. 01 ). Conclusions The analytical performance of two cTnl assays is "clinical accepted" ,the other one is accepted according to guideline. All of them could detect cTnI in apparently healthy subjects. There exist differences among three assays, but their diagnostic characteristics for AMI are not significantly different.
8.The analytical performance assessment of high-sensitivity cardiac troponin T reference intervals investigation
Lingyan SONG ; Jiong WU ; Binbin SONG ; Wenqi SHAO ; Chunyan ZHANG ; Beili WANG ; Wei GUO ; Baishen PAN
Chinese Journal of Laboratory Medicine 2010;33(9):814-818
Objective To assess the analytical performance of hs-cTnT and biological variations in healthy population as well as establish hs-cTnT reference intervals. Methods The serum samples from 100 acute myocardial infraction patients and 474 apparently healthy subjects were collected. The functional sensitivity,within- and between-run imprecision were determined. The hs-cTnT assay and con-cTnT assay were evaluated. The serum hs-cTnT levels were detected in apparently healthy subjects to establish reference intervals. Moreover,the long-term and short-term biological variations for hs-cTnT in healthy volunteers were assessed. Results The functional sensitivity of hs-cTnT was 0. 005 μg/L. The within- and between-run precision for lower level control(0. 014 μg/L) and higher level control(2. 500 μg/L) was 2. 97% vs 3. 64%and 0. 66% vs 1.01% ,respectively. The correlation between hs-cTnT assay and con-cTnT assay was good ( R2 =0. 972 ,P <0. 01 ). The 99th percentile in apparently healthy subjects was 0. 003 μg/L for women less than60 years, 0.008 μg/L for men less than 60 years, 0.015 μg/L for women above 60 years and 0. 021 μg/L for men above 60 years. The CVa, CVi, CVg and CVt of short-term biological variations in detecting hs-cTnT from 22 apparently healthy subjects were 3.8%, 4. 8%, 49.9% and 58.5%,respectively. The CVa,CVi ,CVg and CVt of long-term biological variations were 5. 3% ,6. 4% ,56. 6% and 68. 3% respectively. Conclusions The analytical performance of the hs-cTnT is better than con-cTnT assay,achieving acceptable level according to guideline. Our experimental result could provide the basis for the new high sensitivity cTnT assay in the diagnosis of acute myocardial infarction.
9.Development of a LC-MS/MS method for determination of plasma aldosterone and its performance evaluation
Jiaqian QIN ; Fangjun CHEN ; Yingfei PENG ; Jiong WU ; Chunyan ZHANG ; Beili WANG ; Wei GUO ; Baishen PAN
Chinese Journal of Laboratory Medicine 2017;40(4):247-252
Objective We are going to establish a robust liquid chromatography-tandem mass spectrometric(LC-MS/MS) method for plasma aldosterone assay.Methods 324 healthy individuals were enrolled in Zhongshan Hospital from February to April in 2016 for reference interval survey.The signallinearity,lower limits of quantitation,precision and accuracy of LC-MS/MS have been evaluated.Results from LC-MS/MS and RIA methods were compared.Software SPSS17.0 software was used for statistical analysis.Results The performance characteristics for the method in terms of linearity,lowerlimits of quantitation,precision and accuracy were verified.Linear range of ALD were between 25-2000 pg/ml;the LC-MS/MS assay had a limit of quantitation of 20 pg/ml for ALD;the intra-and inter-assay CV of ALD were <10% and <6%,respectively;the recovery of ALD from serum samples ranged between 97.3 and 105.8% The reference value of ALD in health people ranged between 21-211.6 pg/ml The regression equation by LC-MS/MS (X) and RIA (Y) was:Y =0.271X + 138.900(r=0.43;n=322).Conclusion LC-MS/MS method is robust and reliable for the analysis of aldosterone in plasma and suitable for clinical application.
10.The significance and risk factor analysis of homocysteine in diabetic patients with macroangiopathy
Huifang MO ; Ailun ZHANG ; Jiong WU ; Beili WANG ; Chunyan ZHANG ; Wei GUO ; Baishen PAN
Chinese Journal of Laboratory Medicine 2017;40(7):526-531
Objective To investigate the significance of serum homocysteine (HCY) level in the patients with diabetic macroangiopathy, and analyze the related risk factors.Methods Case control study.279 diabetics (male 198, female 111) aged 59.6(55.0-67.0) were selected in Shanghai Zhongshan Hospital from May 2015 to February 2016.According to the medical history and Carotid intima-media thickness, they were divided into carotid artery disease group (137 cases), cardiovascular disease group (197 cases) and cerebrovascular disease group (29 cases).We detected veinal blood HCY , fasting blood glucose, glycated albumin, total bilirubin, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, L-, gamma glutamyl transferase, urea nitrogen, creatinine, uric acid, cholesterol, three triglyceride, high density lipoprotein cholesterol, low density lipoprotein cholesterol, glycosylated hemoglobin and albumin , creatinine in urine.The groups were compared with Mann-Whitney U test and χ2 test;Pearson correlation analysis was used to determine the correlations between HCY and other indicators;logistic regression model was used to analyze the risk factors of diabetic macroangiopathy and its subclasses;ROC curve was used to analyze the diagnostic value of HCY and uric acid in diabetic macroangiopathy.Results HCY in diabetic with macroangiopathy group was significant hiher than that in diabetic without macroangiopathy group 10.40(8.50-12.48) μmol/L 9.10(7.50-10.70) μmol/L, P<0.01).The incidence of diabetic macroangiopathy (χ2=7.030, P=0.030) and carotid artery lesions (χ2=7.258, P=0.027) was different in patients with different HCY levels.The correlation coefficients of HCY with urea nitrogen, creatinine, uric acid, urinary albumin/creatinine and estimated glomerular filtration rate (eGFR) were 0.340, 0.248, 0.278, 0.133,-0.369 (P<0.05), respectively.HCY was a risk factor for diabetic macroangiopathy, carotid plaque and cardiovascular disease;HCY, age and uric acid were independent risk factors for some of the diabetic macroangiopathy (P<0.05);HCY and UA had a certain diagnostic value for diabetic macroangiopathy(P<0.05).Conclusions Serum HCY is a risk factor for diabetic macroangiopathy, and detection of HCY levels will contribute to the diagnosis and prevention of the disease.