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 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 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 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 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.

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.

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.