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.

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.

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.

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.

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 explore the correlation between sex hormone binding globulin (SHBG) and cardiovascular disease (CVD) in community elderly population.Methods In 2014,1916 elderly people (796 males,and 1 120 females) were selected from Baoshan District Friendship Community,Shanghai.We collected basic epidemiological data and fasting venous blood samples to carry out the detection of biomarkers,and then calculated their ten-year Framingham risk score.In this study,obesity,systolic blood pressure,fasting blood glucose,lipid concentration,and high-sensitive C-reactive protein were considered as CVD risk factors;Framingham risk score was considered as a CVD event prediction risk score.We analyzed the correlations of these factors with SHBG.Results SHBG mean values in the population with a history of CVD were lower than those without a history of CVD (P＜0.001).The correlation coefficient between male SHBG and waist circumference,hip circumference,BMI,systolic pressure,cholesterol,triglycerides,high density lipoprotein cholesterol,apolipoprotein A,high sensitive C-reactive protein were-0.312,-0.307,-0.266,-0.113,0.155,-0.277,0.510,0.394 and-0.130,respectively (P＜0.01).The correlation coefficient between female SHBG and waist circumference,hip circumference,BMI,fasting glucose,cholesterol,triglycerides,high density lipoprotein cholesterol,apolipoprotein A,high-sensitive C-reactive protein were-0.236,-0.248,-0.168,-0.183,0.135,-0.264,0.445,0.358 and-0.295,respectively (P＜0.001).The decrease of SHBG level was consistent with the increase of Framingham score (κ =0.062,P＜0.001).Elevated level of SHBG would reduce the risk of CVD in ten years (P＜0.01).Conclusions There was a negative correlation between baseline SHBG level and CVD risk factors,positive correlation between baseline SHBG level and CVD protection factors in community elderly population;lower SHBG level indicated higher risk of developing CVD events.

Objective To establish the reference interval for CA 72-4 in indirect method.Methods All results for CA72-4 that were stored in our laboratory information system of Zhongshan hospital between Jan.2010 and Dec.2012 were included in this study.Outliers were identified and omitted using Stem-and-Leaf&Box Plots in SPSS statistical software.The treated data was divided into several groups according to gender and age.Nonparametric rank sum test was used to observe the difference between male and female participants and Spearman correlation analysis was used to examine the correlation between CA 72-4 and age.Nonparametric reference intervals for CA 72-4 were estimated statistically in two gender sub-groups.Results After 139 cases excluded, there were 1 548 cases of male (Median 1.7, 0.4 to 18.9) and 773 cases of female ( Median 1.8, 0.2 to 18.9 ).There was a significant difference in serum CA 72-4 between male and female participants.No significant difference was found in serum CA 72-4 among age sub-groups.Indirect reference values for CA 72-4 of male and female were respectively 0 to 8.9 U/ml and 0 to 11.6 U/ml.Conclusion Indirect method to establish biological reference interval is a relatively simple and less expensive method under the high rapid development of the hospital information network .It can be used in the periodical review and establishing the reference intervals where the direct method can not be used.

Objective To set quality goals of conventional biochemical tests through the research of biological variation of the 32 routine items in Chinese population to provide the basis for Chinese clinical and laboratory standards.Methods According to the experimental designs and computing methods from foreign counterparts,the results of biological variation,individual indexes and quality goals were calculated through the serum detection of 22 subjects from clinical laboratory of Zhongshan Hospital in Shanghai (male 12,female 10,ages varying from 20 to 40 years old,median age 30) in short-term (five blood draws within one day at 8:00,10:00,12:00,14:00 and 16:00) and long-term (one blood draw at 8:00 in 6 weeks consecutively) and serum controls (mixed from healthy people).Results (1) Based on the results of shortterm and long-term biological variation in 32 routine itens,the individual indexes and quality goals were obtained.(2)The influence of diet on the biological variation of part of the test items could be observed,especially free fatty acid (the mean value of post-meal was less than pre-meal about 30％),and then followed by high-sensitivity C-reactive protein (the mean value of post-meal was lower than pre-meal about 20％) and triglyceride (the mean value of post-meal was higher than pre-meal about 10％).(3)There were some differences between the quality goals we accessed and the the indicators from Europe and CLIA.Conclusions (1)The results of apolipoprotein E and free fatty acid in this study made up for the inadequate of the European biology database.(2) Only a small part of the 32 routine items were affected by dietary factors.(3) Most quality goals obtained from this study generally consisted with Europe biology quality goals,but a few items existed different.(4)It's more practical and effective to use the results of biological variatiou than CLIA standards for setting up quality goals.

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.

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.