Table of Content

30 November 2016, Volume 31 Issue 11

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Orginal Article

Clinical and laboratory determination analysis of idiopathic cytopenia with uncertain（undetermined） significance

TAO Chaoxin, XING Jiangtao, ZHANG Yuna, LIU Jiangchao, GUO Ligai, XU Liya, ZHU Yun

2016, 31(11):  929-935.  DOI: 10.3969/j.issn.1673-8640.2016.011.001

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Objective To investigate idiopathic cytopenia with uncertain（undetermined） significance（ICUS） and its development. Methods ICUS patients were observed for their bone marrow smears and peripheral blood smears. CD34 antigen,bone marrow biopsy,karyotype analysis and fusion gene WT1 were also determined. Results The durations of 15 ICUS patients were >8 months,and they showed a series of blood cell reducing. There were 11 cases diagnosed in Hebei Medical University Ping'an Hospital. The 2 cases of them had been diagnosed as idiopathic thrombocytopenic purpura in other hospitals,and then they were diagnosed as ICUS because of white blood cell（WBC） and hemoglobin（Hb） reducing later. The other 4 cases were treated in Hebei Medical University Ping'an Hospital after being diagnosed in other hospitals. Some of the 15 patients' bone marrow showed occasional dyshaematopoiesis in erythroid normoblast. Some patients' primitive cells,CD34 antigen and fusion gene WT1 were higher than related reference ranges. Conclusions Various diseases can lead to blood cell reducing. Combined with clinical manifestations and laboratory examinations,whether ICUS will have progress to typical myelodysplastic syndrome（MDS）,aplastic anemia（AA）,or other blood cell reducing diseases,it still requires dynamic clinical observation.

Negative interference by calcium dobesilate in enzymatic assays of free fatty acid

HOU Li'an, GUO Xiuzhi, QIU Ling, CHENG Xinqi, ZHANG Bo, YU Songlin, FANG Huiling, ZHAO Fang, LIU Qian

2016, 31(11):  936-940.  DOI: 10.3969/j.issn.1673-8640.2016.011.002

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Objective To investigate the interference of calcium dobesilate in the determination of free fatty acid（FFA） using 6 acetyl-coenzyme A synthetase （ACS）-peroxidase coupled assays and its influence. Methods Calcium dobesilate solution with different concentrations was prepared by pure water,and was added into sera with 2 different FFA concentrations（550 and 1 200 μmol/L）. Serum FFA levels with final concentrations of calcium dobesilate additions（0,2,4,8,16,32 and 64 μg/mL） were determined using 6 ACS enzymatic assays（A,B,C,D,E and F,randomly）. Bias（%） was calculated in serum FFA with final concentration of calcium dobesilate addition（0 μg/mL）. Taking ±5% as an acceptable range of interference,the changes in serum calcium dobesilate levels observed before and after calcium dobesilate administration [baseline,0 h（valley concentration of drug in the stable state）,1 h,2 h,3 h,4 h and 6 h] in 40 participants were monitored by high performance liquid chromatography（HPLC）. Serum calcium dobesilate levels in 40 participants from those taking calcium dobesilate were determined. Results The level of serum calcium dobesilate（0 h） [median（interquartile range）] was 6.64（5.64-7.86）μg/mL. After taking 500 mg,the level of serum calcium dobesilate peaked at 2-3 h. The peak level of serum calcium dobesilate was 15.00（13.63-19.67）μg/mL,and then decreased gradually. The average level in 40 participants was 11.92（5.45-19.22）μg/mL. With the increase of serum calcium dobesilate level,the degree of interference in FFA determination also increased in in vitro experiment. For the low and high concentrations（550 and 1 200 μmol/L） of serum FFA,the biases in 6 ACS enzymatic assays were -0.40% - -65.01% and-0.20% - -52.33% in 8 μg/mL serum calcium dobesilate. The interference degrees of 4 ACS enzymatic assays can be accepted,except B and D. The biases in 6 ACS enzymatic assays were -1.68% - -28.28% and -1.81% - -16.48% in 16 μg/mL serum calcium dobesilate. Except kit A and kit C（high concentration of FFA）,the interference of other kits and kit C（low concentration of FFA） was beyond the acceptable range. The degree of interference was also related with basic serum FFA concentration. Under same calcium dobesilate concentration,the interference with lower concentration of serum FFA was more obvious. Conclusions In ACS enzymatic assays,calcium dobesilate produces a negative interference in the determination of FFA.

Relationships of homocysteine,blood lipids and D-dimer with folic acid intervention in patients with acute cerebral stroke

CANG Shenyuan

2016, 31(11):  941-943.  DOI: 10.3969/j.issn.1673-8640.2016.011.003

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Objective To investigate the relationships of homocysteine（Hcy）,4 items of blood lipids [total cholesterol（TC）,triglyceride（TG）,low-density lipoprotein cholesterol（LDL-C） and high-density lipoprotein cholesterol（HDL-C）] and D-dimer（DD） with folic acid intervention in patients with acute cerebral stroke. Methods A total of 93 patients with acute cerebral stroke were enrolled. According to random number table,they were classified randomly into observation group（47 cases） and control group（46 cases）. Control group was treated with conventional therapy,and observation group was treated with conventional therapy combined with folic acid treatment. The 2 therapies lasted 4 weeks. The levels of Hcy,4 items of blood lipids and DD of the 2 groups were compared. Results Before therapies,plasma Hcy,TC,TG,LDL-C,HDL-C and DD levels showed no statistical significance between the 2 groups（P>0.05）. Plasma Hcy,TC,TG,LDL-C and DD levels in observation group were lower than those in control group after therapies（P<0.05）,and HDL-C level was higher than that in control group（P<0.05）. Conclusions Folic acid can reduce DD and Hcy levels in patients with acute cerebral stroke,which can improve patients' status of dyslipidemia.

Comparison of dried blood spot samples and serum samples for HBV DNA quantification determination and drug resistance mutation

ZHANG Min, WANG Qianying, LI Xin, WANG Gang, HU Yunwen

2016, 31(11):  944-947.  DOI: 10.3969/j.issn.1673-8640.2016.011.004

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Objective To assess the feasibility of dried blood spot（DBS） sample as an alternative to serum sample for hepatitis B virus（HBV）determination. Methods DBS samples and serum samples were prepared through extracting venous blood,and HBV DNA was determined for 100 patients with chronic hepatitis B,whose HBV virus load >1×10² copies/mL. DBS and serum sample results were compared by correlation analysis. HBV genotypes were analyzed in 73 patients of genotype B and genotype C. A total of 56 DBS and serum samples from patients with hepatitis B were collected randomly. Using an in-house nest polymerase chain reaction（PCR） for reverse transcriptase segment,the data were analyzed using geno2pheno. Results HBV DNA of DBS samples was determined in a range of 2-8 log₁₀ copies/mL. The correlation coefficient（r²） for HBV DNA between DBS samples and serum samples was 0.82（P<0.05）. The quantification of HBV DNA determination in DBS samples was 1 log₁₀ lower than that in serum samples. The coincidence rate of the 2 kinds of samples was 95.83%. For DBS and serum samples,the determination rates for drug resistance were 82.14% and 85.71%（P>0.05）. The coincidence rate of DBS and serum samples for HBV genotype was 100.00%. Conclusions DBS samples can be used in HBV DNA quantification and drug resistance determinations.

Correlation between serum HBcrAg and intracellular HBV DNA in patients with hepatocellular carcinoma

LI Huiming, JIA Jian'an, WANG Mengmeng, GU Xing, FANG Meng, GAO Chunfang

2016, 31(11):  948-952.  DOI: 10.3969/j.issn.1673-8640.2016.011.005

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Objective To assess the correlations of a newly-developed hepatitis B virus（HBV） infection marker,hepatitis B core-related antigen（HBcrAg）,with covalent closed circular DNA（cccDNA） and HBV DNA in tumor tissue and adjacent non-tumor tissue among hepatocellular carcinoma（HCC） patients. Methods A total of 95 HBV infection-related patients who received hepatic resection for HCC were enrolled. Serum HBcrAg were determined by chemiluminescence immunoassay. HBV DNA and cccDNA in tumor tissue and adjacent non-tumor tissue were quantified by polymerase chain reaction（PCR） fluorescent probe. Results The levels of HBcrAg in hepatitis B e antigen（HBeAg）⁺/hepatitis B e antibody（HBeAb）^- group,HBeAg⁺/HBeAb⁺ group and HBeAg^-/HBeAb⁺ group were （6.41±1.18）,（5.80±0.50） and（5.16±0.94）lgU/mL,respectively,and there was statistical significance among these 3 groups（P<0.001）. There were correlations of HBcrAg with adjacent non-tumor tissue cccDNA and adjacent non-tumor tissue HBV DNA in HCC patients（r=0.359 and 0.440,P<0.001）. In HCC patients of TNM stage Ⅰ,HBcrAg was associated with adjacent non-tumor tissue cccDNA and adjacent non-tumor tissue HBV DNA（r=0.491 and 0.578,P<0.01）. In HCC patients with TNM stage Ⅱ,HBcrAg was correlated with adjacent non-tumor tissue cccDNA and adjacent non-tumor tissue HBV DNA（r=0.339 and 0.402,P<0.05）. However, in HCC patients with TNM stage Ⅲ,no correlation of HBcrAg and adjacent non-tumor tissue cccDNA and adjacent non-tumor tissue HBV DNA. Conclusions HBcrAg can help with distinguishing HBeAg positive and negative HCC patients. Although HBcrAg has correlations with adjacent non-tumor tissue HBV DNA and cccDNA in hepatitis B surface antigen（HBsAg）⁺ HCC patients,it does not show better correlations than other serum markers such as HBeAg and HBV DNA.

Etiological analysis of infectious diarrhea in Wuxi,2014

GUAN Hongxia, XIAO Yong, SHA Dan

2016, 31(11):  953-958.  DOI: 10.3969/j.issn.1673-8640.2016.011.006

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Objective To understand the pathogen spectrum and epidemiological characteristics of infectious diarrhea in Wuxi. Methods The stool specimens from outpatients with diarrhea were collected from sentinel hospitals from January to December 2014. The nucleic acid of norovirus,rotavirus,enteric adenovirus,human astrovirus and sapovirus was determined by real-time fluorescence polymerase chain reaction（PCR）. Diarrheagenic Escherichia coli,Salmonella spp,Shigella spp,Vibrio parahaemolyticus and Vibrio cholerae were identified by conventional culture methods. Pathogenic composition,seasonal variation and population distribution were analyzed. Results A total of 915 specimens were monitored. The positive rate of viral nucleic acid was 14.4%,and norovirus（9.4%） and rotavirus（3.4%） showed obvious seasonal distribution characteristics with the peak incidence occurring in winter and spring. The infection rate of children of 0-5 years old was higher than those of the other age groups. Bacteria,with the positive rate 15.0%,were isolated from June to September. Salmonella spp（6.0%） was the main isolate whose predominant serotypes were Salmonella enteritidis and Salmonella typhimurium,and the second was diarrheagenic Escherichia coli（4.6%）. Conclusions The viral diarrhea deserves attention in winter and spring,norovirus and rotavirus are the common pathogens. The bacterial diarrhea is given priority in summer,whose dominant pathogens are Salmonella spp and diarrheagenic Escherichia coli. Children under 5 years old are main for the prevention and control of viral diarrhea.

Drug resistance of ESBLs-producing Escherichia coli to aminoglycosides and the sequences of drug resistant genes

WU Rong, LÜ Yujiang, DAI Junhua, XIANG Fenfen, KONG Qianqian, QIAN Ning, ZHANG Guiliu, YANG Yuling, CHEN Lichun, KANG Xiangdong

2016, 31(11):  959-964.  DOI: 10.3969/j.issn.1673-8640.2016.011.007

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Objective To study the drug resistance of extended-spectrum beta-lactamases（ESBLs）-producing Escherichia coli to aminoglycosides and the expression frequencies and sequences of drug resistant genes in Jiangchuan District,Yuxi City,Yunnan Province. Methods The drug resistance of 32 isolates of ESBLs-producing Escherichia coli to gentamycin,amikacin,kanamycin,tobramycin and netilmicin was determined by disk diffusion test. A total of 7 aminoglycoside-modifying enzyme（AME） genes [aac（3）-Ⅰ,aac（3）-Ⅱ,aac（6′）-Ⅰad,aac（6′）-Ⅰb,aac（6′）-Ⅱ,ant（3″）-Ⅰ and ant（2″）-Ⅰ] and 6 genes of 16S rRNA methylases（armA,rmtA,rmtB,rmtC,rmtD and npmA） were determined by polymerase chain reaction（PCR）,and the sequence of aac（3）-Ⅱ gene was analyzed by double deoxidizing terminal cessation method. Results The drug resistant rates of 32 isolates of ESBLs-producing Escherichia coli to gentamycin,amikacin,netilmicin,kanamycin and tobramycin were 100%,0%,0%,18% and 87%,respectively. It was found that 2 AME genes existed simultaneously in 6 isolates. The positive rates of 4 AME genes,aac（3）-Ⅱ,aac（6′）-Ⅰb,ant（3″）-Ⅰ and ant（2″）-Ⅰ,were 100%,9.4%,6.2% and 3.1% in 32 isolates. There was no gene of 16S rRNA methylases. The aac（3）-Ⅱ mutations existed in 4 out of 32 isolates,which were G232A,T251C,G580A and T612A,respectively. There was no gene of AME and 16S rRNA methylases in 8 isolates of ESBLs-producing Escherichia coli. Conclusions The drug resistance of ESBLs-producingEscherichia coli to aminoglycosides was correlated with AME gene expressions,and the mutations of aac（3）-Ⅱ may contribute to the increased drug resistance to netilmicin.

Consistency of 2 systems for the determination of antinuclear antibody profile

ZHAN Wenli, LI Lixin, HUANG Zhuochun, HU Jing, WU Yongkang

2016, 31(11):  969-973.  DOI: 10.3969/j.issn.1673-8640.2016.011.010

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Objective To evaluate the consistency of commonly-used 2 systems for the determination of antinuclear antibody（ANA） profile,and to provide a reference for choosing methods for ANA profile determination. Methods Sera from 358 patients with systemic lupus erythematosus（SLE）,147 patients with autoimmune disease and 47 healthy subjects were collected,and were determined for ANA profile by 2 systems,HUMAN（Germany） Diagnostics Ltd. and EUROIMMUN（China） Medical Diagnosis Ltd.. The calculated consistency and McNemar test were used to evaluate the consistency of 2 systems. Results The consistencies of anti-Smith（Sm） antibody,anti-ribosomal protein（RIB） antibody and anti-ribonucleoprotein（RNP） antibody were 79.9%,93.5% and 82.4%,with statistical significance（P<0.001）. The consistencies of other 4 indices[anti-Sjogren syndrome A（SS-A） antibody,anti-Sjogren syndrome B（SS-B）antibody,anti-scleroderma 70（Scl-70） antibody and anti-Jo-1 antibody] were >90%,without statistical significance（P>0.05）. Among 358 SLE patients,the positive rates of anti-Sm antibody were 48.6% by HUMAN and 27.1% by EUROIMMUN. Among 194 non-SLE patients,the specificities of anti-Sm antibody were 93.3% by HUMAN and 99.5% by EUROIMMUN.Conclusions The consistencies of HUMAN and EUROIMMUN in determining anti-Sm,anti-RIB and anti-RNP antibodies are low,while the other 4 indices show good consistencies. For the determination of anti-Sm antibody,HUMAN reveals a better determination rate and lower specificity than EUROIMMUN.

Evaluation on the detection methods of group B Streptococcus in late pregnancy

ZHANG Jinghua, YUAN Yinghua, CHENG Jie, SUN Fenyong

2016, 31(11):  974-977.  DOI: 10.3969/j.issn.1673-8640.2016.011.011

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Objective To evaluate the detection methods of group B Streptococcus（GBS） in late pregnancy. Methods A total of 300 cases of prenatal genital tract secretion samples in late pregnancy were detected for GBS by broth enrichment switched to blood plate method,screening for GBS cultivation medium method and polymerase chain reaction（PCR）. Methodological evaluation was performed. Results The detection rates of GBS by broth enrichment switched to blood plate method and screening for GBS cultivation medium method were 5.3% and 7.0%,without statistical significance（P>0.05）. The detection rate of GBS by PCR was 9.6%,and there was statistical significance with the other 2 methods（P<0.05）. Conclusions PCR is a good choice for GBS detection in late pregnancy. It can be used in the rapid detection of GBS for pregnant women before delivery．

MiR-125b increasing the susceptibility of hepatic cancer stem cells to doxorubicin through down-regulating the expression of MCL-1

HONG Dongcheng, ZHANG Benhong, GONG Binbin

2016, 31(11):  981-986.  DOI: 10.3969/j.issn.1673-8640.2016.011.013

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Objective To investigate the role of microRNA-125b（miR-125b）increasing the killing activity of doxorubicin to hepatic cancer HepG2 stem cells and its mechanism. Methods HepG2 cells treated with miR-125b,doxorubicin and myeloid cell leukemia-1（MCL-1）plasmid. Flow cytometry was used to determine the proportions of HepG2 cells,3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide（MTT） method was used to determine cell vitalities,and cell apoptosis rate was determined by Annexin V staining method. Cell mitochondrial membrane potential was determined by JC-1 dyeing method,and western blotting was used to determine the target protein [MCL-1,cysteine aspartic acid specific protease-9（caspase-9） and cysteine aspartic acid specific protease-3（caspase-3）]. Results Single treatment of doxorubicin increased the proportion of HepG2 cells,however,the combined treatment with doxorubicin and miR-125b decreased it. The results of MTT method and flow cytometry showed that miR-125b could enrich the killing activity and cell apoptosis of doxorubicin to HepG2 cells. The results of JC-1 dyeing method showed that miR-125b enhanced the damage of mitochondria induced by doxorubicin. The results of western blotting indicated that miR-125b increased the activation of caspase-9 and caspase-3 in doxorubicin-treated HepG2 cells. MCL-1 plasmid impaired the synergistic effect of miR-125b on doxorubicin-induced cell death and apoptosis in HepG2 cells. Conclusions miR-125b increased the susceptibility of HepG2 cells to doxorubicin through down-regulating the expression of MCL-1.

Transcription factor Flo8 G723R and T751D mutations enhancing virulence in Candida albicans

LI Wenjing, SHEN Yun, LIU Jinyan, SHI Ce, WANG Ying, ZHAO Yue, XIANG Mingjie

2016, 31(11):  987-992.  DOI: 10.3969/j.issn.1673-8640.2016.011.014

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Objective To study the relationship between Candida albicans transcription factor Flo8 G723R and T751D mutations and their virulence. Methods The lethality rate,renal fungal burden of infected mice and the expression of genes for virulence factors in Candida albicans Flo8 wild-type SN152,Flo8 complementation strain Flo8^SN152,Flo8 mutation strains Flo8^G723R and Flo8^T751D were compared. The relationship between Flo8 mutations and Candida albicans virulence was analyzed. Results Compared to SN152 and Flo8^SN152,Flo8 mutation strains Flo8^G723R and Flo8^T751D produced a higher lethality rate and more renal fungal burden of infected mice and the expression of genes for virulence factors,and increased at different degrees. Conclusions Transcription factor Flo8 G723R and T751D mutations increase the virulence and the expression of genes for partial virulence factors in Candida albicans.

Application of Westgard sigma rules in selecting internal quality control rules for clinical hematology tests

FEI Yang, WANG Wei, WANG Zhiguo

2016, 31(11):  993-996.  DOI: 10.3969/j.issn.1673-8640.2016.011.015

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Objective To use Westgard sigma rules in selecting suitable internal quality control rules for clinical hematology tests. Methods A laboratory which participated complete blood count external quality assessment and internal quality control of the National Center for Clinical Laboratories in 2013 was enrolled. Accumulated coefficient of variation（CV） in internal quality control was regarded as the estimation value of imprecision,and the percentage difference in external quality assessment was chosen to be the estimation value of bias. The allowable total errors（TEa） based on biological variation, the Clinical Laboratory Improvement Amendments of 1988（CLIA'88） and national health industry standard WS/T406-2012 were adopted as quality specification. σ value was calculated,and suitable internal quality control rules were selected by Westgard sigma rules. Results When using TEa based on biological variation,1_3s/2_2s/R_4s/4_1s/8_x multi-rules were selected for all complete blood counts whose σ values were < 4. If TEa in CLIA'88 was employed,1_3s/2_2s/R_4s multi-rules were selected for hemoglobin and platelet whose σ values were 5.20 and 5.13. When TEa in national health industry standard was used,1_3s/2_2s/R_4s/4_1s multi-rules were suitable for hemoglobin and hematocrit whose σ values were 4.35 and 4.62. Conclusions Westgard sigma rules are convenient and feasible,and laboratories can use them for selecting internal quality control rules correctly.

Research progress of the relationship between microRNA and platelet

CHEN Shuying, SHI Chaohui, LIN Yong

2016, 31(11):  997-1001.  DOI: 10.3969/j.issn.1673-8640.2016.011.016

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MicroRNA（miRNA） is 22-nucleotide noncoding single-stranded RNA. Its main function is to regulate gene expression after transcription,so as to participate in regulating biologic activity. Though platelet has no cell nucleus,it plays an important role in hemostasis and coagulation processes. Understanding the role of platelet production and activity deeply will provide new ideas for the treatment of platelet-related blood systemic diseases.

CX3CL1 and CX3CR1 in lesion mechanism of atherosclerotic heart diseases

LI Yongshu, ZHANG Baohuan, JIA Kegang, LIU Junfeng

2016, 31(11):  1002-1010.  DOI: 10.3969/j.issn.1673-8640.2016.011.017

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Fractalkine（CX3CL1） and its specific receptor CX3C chemokine receptor1（CX3CR1）,CX3CL1-CX3CR1 axis are all involved in the formation and development of atherosclerosis,which change the plaque composition and stability of plaques. CX3CR1 V249I and CX3CR1 T280M are associated with coronary artery lesions. In atherosclerotic plaque,CX3CL1 and CX3CR1 were expressed by vascular smooth muscle cells（VSMC） and monocytes/macrophages. Interaction between vascular smooth muscle cells（VSMC） and monocyte needs CX3CL1-CX3CR1 axis,and this mutual effect regulates the survival and differentiation of monocyte. Through a series of mechanism,such as involved nuclear factor-kappa B（NF-κB）,activated protein-1（AP-1） and signal transducers and activators of transcription（STAT） 1/STAT3,resistin up-regulates the expressions of CX3CL1 and CX3CR1,and generates pro-inflammatory state of smooth muscle cells. Serum CX3CL1 level is increased in patients with unstable coronary artery diseases and patients with severe coronary artery lesions. Serum CX3CR1 level is increased in patients with coronary artery stenosis,which is not associated with the degree of coronary artery stenosis. The related study on CX3CL1/CX3CR1 shows the potential mechanism and pathogenicity effect of some inflammatory mediators in atherosclerotic heart diseases,so as to provide a reference for clinical treatment strategies and drug intervention studies.