Objective To preparing the reference material(RM) of lactate dehydrogenase(LD),we purification of LD from human erythrocyte(RBC)and studied its properties.Methods Using a modified procedure which including complete hemolysis of the RBC,hydroxyapatite treatment,(NH4)2 SO 4 Precipitation,CM- Sephadex C 50,Sephadex G100 and 5'- AMP- Sepharose 4B affinity chromatography.Results The purified LD has a sepecific activity of 163.0 kU/g protein.It is almost free of contaminating enzymes.Two corresponding bands were observed on both PAG plates stained for either protein or LD activity after electrophresis had been done.The apparent Michaelis constants were of 1.000 and 0.179 mmol/L for L-lactate and NAD and of 0.119 mmol/L 0.062 mmol/L for pyruvate and NADH respectively.Conclusion The final purified LD was found to be very similar to that in human serum in catalytic properties.It is intended to be a fundament to prepare a RM for the measurement of LD.

Objective To study on glycogen assay,polymerae chain reaction,and cell culture for diagnostic value of chlamydia infection of vervical smeat.Methods 106 specimens were examined by using glycogen assay,PCR and cell culture.Results Compared with cell culture,the sensitivity and specifity of glycogen assay are 80.0％ and 95.8％ ,and the sensitivity and specifity of PCR are 90.0％ and 97.9％ ,respectively.Conclusion The glycogen assay possesses diagnostic value for chlamydia trachomatis infection of vervical smear.

Objective To evaluate the feasibility of PerkinElmer Genetic Screening Processor(GSP) in the application of newborn screening for congenital hypothyroidism (CH) and congenital adrenal hyperplasia (CAH) by detecting thyroid-stimulating hormone (TSH) and 17-OH-progesterone(17-OHP).Methods The dried-blood spots specimens from Centers for Disease Control and Prevention(CDC) and the quality control in the reagent kit were detected and the accuracy,precision and linearity were calculated.A total of 1 012 samples of TSH(60 of positive and 952 negative samples) and 991 samples of 17-0HP(34 positive and 957 negative samples)were detected.The initial cut-off value was determined by ROC curve determined.The consistency between the results from GSP and clinical diagnosis was analyzed.Results The average of within-run coefficient of variation(CV) of TSH and 17-OHP were 6.69％ to 12.6％ and 7.52％ to 9.29％,and the average of between-run CV were 6.91％ to 10.96％ and 6.86％ to 12.36％,respectively.The average of bias of TSH and 170HP were-14.28％ to-0.74％ and-0.45％ to 12.54％.The linearity of GSP detection was fine.The initial cut-off values were 23.43 U/mL(TSH) and 21.42 ng/mL(17-OHP).The sensitivity of GSP detection was 100％ and the specificity of TSH and 17-OHP were 98.11％ and 99.58 ％ respectively.The results of GSP detection showed good consistency with clinical diagnosis.Conclusion As the first real automatic fluorescence immunoassay analyzer,GSP could be used in routine clinical diagnosis for CH and CAH.

Objective To select the calibrator for the conventional measurement system of serum a-Amylase (Amy).Methods The Amy levels of forty frozen serum samples were detected by the IFCC reference method (reference system),the conventional system A which used the Bioassay routine reagent and Randox calibrator,and was calibrated by the Roche PNPG7 method,and the conventional system B which used the Bioassay routine reagent and Randox calibrator,and was calibrated by the Rondox liquid stable PNPG7 method,respectively,and the acceptability of the two conventional systems was evaluated.Results The regression equations of the measurement values between the IFCC reference method and the conventional systems A and B were Y =0.964X +0.376 and Y =1.095X + 3.131,respectively.Among them,X and Y represented the results of the IFCC reference method and the conventional system,respectively.Compared with the IFCC reference method,the results of the conventional system A was reliable.Condusion With the guidance of the IFCC reference method,the domestic biochemical reagents matched with the suitable calibrators may provide the acceptable results.

Objective To improve the protective strategy of occupational risks for technicians in blood centers so as to ensure their health and safety.Methods The occupational risk factors were analyzed by the 5W1H method,and the corresponding protective measures were introduced.Then,the protective measures were verified dynamically by the PDCA circulation,and were standardized and further spread.The projects with poor effect were replanned and entered the PDCA recycling.Results The system of the 5W1H method combined with PDCA circulation could identify and control various occupational risks,and effectively prevent the occupational detriments.Condusion Combined application of the 5W1H method with PDCA circulation is an effective way to protect the health and safety of technicians in blood centers and an effective mode to improve the management of occupational risks.

Objective To evaluate the detection competence of HbA2 and HbF in Guangxi medical laboratories.Methods The external quality assessment(EQA) of HbA2 and HbF was conducted twice a year and five samples was detected each time during 2012 to 2016.The laboratories participated in EQA completed the samples' detection and submitted the detection results at specified time according to the requirements of EQA.The distribution of each detection system,the qualification rate of each laboratory,the variation degrees of each detection system and each detection method,and the variations of results for different levels of quality control(QC) materials during 5 years were analyzed based on the returned results.Results The application of high performance liquid chromatography (HPLC) and capillary electrophoresis(CE) increased year by year and their usage rates in 2016 reached up to 46.1％ (82/178) and 18.0％ (32/178),respectively.The qualification rates of HbA2 and HbF increased from 51.5％ (34/66) and 60.6％ (40/66) in 2012 to 93.3％ (166/178) and 92.1％ (164/178) in 2016,respectively.The average coefficient of variation(CV) of each detection system decreased year by year.There were good CVs for the results of high,medium and low levels of HbA2 QC materials detected by the Bio-Rad Variant Ⅱ and Sebia CAPILLARYS 2 systems,and they were less than 6.0％.HPLC and CE could quantitatively detect the HbA2 and HbF levels,and their total detection competence was superior to that of agarose gel electrophoresis.Conclusion EQA can assess the abihty of one laboratory detecting HbA2 and HbF,and quantitatively analyze the levels of HbA2 and HbF,which may provide the quality assurance and data support for the screening and prevention of thalassemia.

Objective To scan protein expression profile of immune complexes (ICs) derived from the synovial tissue of the patients with rheumatoid arthritis (RA) based on liquid chromatography-tandem mass spectrometry (LC-MS).Methods The samples of synovial fluid were obtained from knee joints of the patients with RA and osteoarthritis (OA) used as control during therapeutic arthrocentesis in knee jiont at the Department of Orthopedics of Jinling Hospital,School of Medicine,Nanjing University.The protein expression profile of ICs was identified by enrichment strategy based on immunoprecipitation and LC-MS analysis.The value of fraction of total (FOT) was used to estimate protein abundance and screen the up-and down-regulated proteins.The function enrichment,interaction network and signal pathway of differential proteins were analyzed using softwares David and String.Results A total of 511 and 526 protein spots in ICs of RA and OA patients were identified respectively.Among them,170 proteins existed only in RA group.45 and 85 proteins in RA group were statistically up-and down-expressed compared with controls.Conclusion HSP90AA1,HSP70,HLAG,Thioredoxin,Annexin A2 and vitronectin may be involved in the pathogenesis of RA through different paths and possible to become promising diagnostic indicators or new therapeutic targets for RA.

Objective To evaluate the application values of two kinds of single-platform flow cytometric methods,the Volumetric method based on flow sensor and the Trucount method based on Trucount beads,in the counts of T-lymphocyte subsets in peripheral blood of patients after transplantation.Methods The absolute number and percentage of CD4 +,CD8 +,and CD3 + T cells in peripheral blood samples from 107 patients after liver or renal transplantation were determined by the Trucount method and the Volumetric method,respectively,and their results were compared using paired t-test and linear regression analysis.Five samples with low CD3 + counts were selected and the precisions of the absolute number of CD4 +,CD8 + and CD3 + T ceils detected by the Volumetric method were evaluated.Results There was no significant difference in the levels of CD4+,CD4+/CD3+,CD8+,CD8+/CD3+,and CD4+/CD8 + in peripheral blood between the Trucount method and the Volumetric method (P ＞ 0.05),and the linear regression coefficients between them were from 0.9 to 1.1.When the concentration of CD3 + was equal or more than 40/μL,the coefficients of variation (CVs) were below 5.5％ for the Volumetric method.When the concentration of CD3 + was 20/μL,the CVs of CD3 +,CD4 +,and CD8 + were 5.19％,10.28％ and 6.48％,respectively.Conclusion The single-platform method based on flow sensor is accurate and reproducible for counting T-lymphocyte subsets in peripheral blood,which may be used to monitor the immune state of the patients after liver or renal transplantation.

Objective To investigate the correlations of Eotaxin-1,rheumatoid factor (RF) and anti-cyclic citrullinated peptide (CCP) antibody with the activity of rheumatoid arthritis (RA).Methods A total of 58 patients with early RA,46 patients without RA and 53 healthy controls from our hospital during December 2014 and December 2015 were enrolled in our study.The activity of RA was evaluated by the swollen joint count (SJC),tender joint count (TJC) and DAS28 score.The levels of serum Eotaxin-1 and antiCCP antibody were detected by ELISA and serum RF levels were determined by the immunological turbidimetry.The comparisons of serum Eotaxin-1,anti-CCP antibody and RF levels between different groups were performed with ANOVA and their correlations with SJC,TJC and DAS28 score were analyzed by Spearman rank correlation.Results The levels of Eotaxin-1,anti-CCP antibody and RF in RA patients were (96.02 ± 2 1.07) pg/mL,(183.42 ± 87.45) U/mL and (119.09 ± 62.30) RU/mL,respectively,which were significantly higher than those in the patients without RA and healthy controls (P ＜ 0.01).The levels of serum Eotaxin-1 in RA patients were significantly related to TJC,SJC and DAS28 score (P ＜ 0.01),while the levels of anti-CCP antibody were related to TJC and DAS28 score.The levels of RF were only related to DAS28 score.Conclusion The levels of serum Eotaxin-1 and anti-CCP antibody in RA patients are significantly correlated with the activity of RA,which may be new serum markers to monitor the activity of RA.

Objective To investigate the values of combined detection of serum pancreas autoantibodies (PAB),anti-saccharomyces cerevisiae antibodies(ASCA),goblet cell autoantibodies(GAB) and antineutrophil cytoplasmic antibodies(PANCA) and fecal calprotectin(FC) in the diagnosis and differential diagnosis of inflammatory bowel diseases(IBD).Methods The serum and feces samples from IBD patients,including 107 with definite Crohn's disease(CD) and 98 with definite ulcerative colitis(UC),and 79 non-IBD patients as the control were collected.Serum PANCA,ASCA,GAB and PAB were detected by an indirect immunofluorescence assay,and FC concentration by double-antibody sandwich ELISA.The results from different patients were compared and analyzed.Results The positive rates of serum PANCA,GAB,PAB and ASCA in 205 IBD patients were 36.1％,29.8％,38.0％ and 4.9％,respectively.The FC concentrations in IBD,CD and UC patients were significantly higher than that in the control(P ＜ 0.01),while there was no statistical difference between CD and UC patients (P ＞ 0.05).The positive rates of PANCA in CD and UC patients were 8.4％ and 66.3％,respectively,while those of PAB in CD and UC patients were 65.4％ and 8.2％,respectively.The sensitivity and specificity of PAB,PANCA,GAB,ASCA,FC and their combination in the differential diagnosis of IBD and non-IBD were 38.0％,36.1％,29.8％,4.9％,54.1％,63.4％ and 98.7％,96.2％,94.9％,100％,68.4％,93.7％,respectively.The area under the ROC of the combination of 5 markers was 0.819 in differentially diagnosing IBD and non-IBD.The area under the ROC of PANCA for the differential diagnosis of UC was 0.816,while that of PAB for the differential diagnosis of CD was 0.823.Conclusion GAB is an autoantibody associated with IBD,which may be helpful for the auxiliary diagnosis of IBD.PAB and PANCA are the important serological markers for the diagnosis of CD and UC,respectively.The combination of FC with PAB,PANCA,GAB and ASCA may be used for the differential diagnosis of IBD and non-IBD,but has little value in distinguishing CD and UC.