Table of Content

30 May 2014, Volume 29 Issue 5

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the Human Genome Project to the Cancer Genome Atlas, Are You Ready?

ZHU Yusheng

2014, 29(5):  409-413.  DOI: 10.3969/j.issn.1673-8640.2014.05.001

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In the last decade, enormous progress has been made in the understanding of mechanism of tumorigenesis as well as in prevention, diagnosis and treatment of tumor because of many breakthroughs in tumor molecular biology research. This editorial introduces the review and original research papers published in this special issue of "Tumor-associated Genes". These articles provide our readers with up-to-date information on human cancer genome, genetic polymorphisms and miRNAs in the field of cancer research. We hope that the new information promotes more studies on tumor-associated genes and more clinical applications of tumor-associated gene tests in the field of laboratory medicine.

The Human Cancer Genome: Laboratory Analysis and Clinical Application

LI Shiyong

2014, 29(5):  414-434.  DOI: 10.3969/j.issn.1673-8640.2014.05.002

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Genomic aberrations cause cancers through activation of cancer genes and consequently functional changes of cellular processes or signal transduction pathways. Technological advances have allowed us to interrogate DNA aberrations not only in a single cancer gene, but also in a panel of cancer genes or the entire human cancer genome. Discovery of new genomic aberrations has increased our understanding of carcinogenesis, and revolutionalized the diagnosis, classification and treatment of human cancers as well. This review provides an overview of the organization of the human genome, laboratory methods of human genome analysis, genomic alterations in human cancers, and clinical application of human cancer genomics.

Relationship between microRNA-related single nucleotide polymorphism and tumors

LIANG Qiuli, LI Shujin, NI Peihua.

2014, 29(5):  435-440.  DOI: 10.3969/j.issn.1673-8640.2014.05.003

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MicroRNAs (miRNAs) are well-conserved small non-coding RNAs, which play roles in transcriptional and post-transcriptional regulation of gene expression. MiRNAs participate in diverse biological pathways and may act as oncogenes or tumor suppressors. Single nucleotide polymorphism(SNP) in miRNAs might promote carcinogenesis and tumor development, which may also affect early diagnosis, prognosis and treatment. Here, we highlight the association between miRNA-related SNP and tumors and describe the miRNA polymorphisms and SNP in miRNA target gene.

Research progress on the correlation of LTA single nucleotide polymorphism with the susceptibility of gastric cancer

WANG Hongling, LU Renquan, GUO Lin.

2014, 29(5):  441-445.  DOI: 10.3969/j.issn.1673-8640.2014.05.004

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Although the morbidity and mortality of gastric cancer is on the decline around the world, it is still rising in China recently and has become one of main factors that influences on the quality of people′s life.The pathogenesis of gastric cancer is associated with various factors, involving in gene polymorphism, inflammatory cytokines, biochemical mechanisms and so on. In particular, lymphotoxin-alpha(LTA) gene single nucleotide polymorphism has been a hot spot in the research of gastric carcinogenesis in recent years.While LTA rs909253(A/G), rs1041981(C/A), rs2229094(T/C), rs746868(G/C)and so on have some associations with gastric cancer, there is no consistent result.This article reviews the current progress in the relationship between LTA gene polymorphism and the susceptibility of gastric cancer.

Modulation of the proliferation, migration and SIRT1 expression of prostate cancer cell PC-3 by miRNA-221 and miRNA-222

YANG Xiao, GAN Rong, YANG Yingmei, ZHAO Lingxu, BO Jinshuang, GUAN Yanming, MENG Qinghe, L Jianxin.

2014, 29(5):  446-451.  DOI: 10.3969/j.issn.1673-8640.2014.05.005

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Objective To investigate the roles of micro RNA(miRNA)-221 and miRNA-222 on the proliferation and migration of prostate cancer cell PC-3 and the influence on silent information regulator 1(SIRT1). Methods The groups were arranged as cells without treatment (control group), cells transfected with pEX-5 empty plasmid (pEX-5 group), cells transfected with miRNA-221 inhibitor (miRNA-221 inhibition group) and cells transfected with miRNA-222 inhibitor (miRNA-222 inhibition group). The influence of miRNA-221 and miRNA-222 on prostate cancer cell proliferation was determined by CCK-8, and the migration ability was determined by wound healing test. The protein and mRNA levels of SIRT1 were determined by Western blotting and fluorescence quantitation polymerase chain reaction (PCR), respectively. The prediction analysis was performed by miRNAanda target. Results After the expression inhibition of miRNA-221 or miRNA-222 in cell PC-3, the cell activities were determined for 7 d. The activities (A_{450 mm}) in miRNA-221 and miRNA-222 inhibition groups were down-regulated about 1.5-2.0 times compared with that in pEX-5 group, and there were significant differences between the 2 groups from the 3rd d(t=6.7, P<0.01; t=5.3, P<0.01). The wound healing test showed that the migration abilities of miRNA-221 inhibition group and miRNA-222 inhibition group decreased than that of pEX-5 group. Moreover, the expression levels of SIRT1 protein in miRNA-221 inhibition group(0.26±0.021) and miRNA-222 inhibition group(0.21±0.005 8) were up-regulated compared with that in pEX-5 group(0.14±0.017), and there were statistical significances (t=6.3 and 6.4, P<0.05 ). The SIRT1 mRNA expression levels had no statistical significance among the 3 groups(P>0.05). Conclusions The miRNA-221 and miRNA-222 promote the proliferation and migration of prostate cancer cell PC-3 and have the influence on SIRT1 expression in protein level.

Inhibition of down-regulated miRNA-221 and miRNA-222 in human breast cancer MCF-7 cell line to tumor cell proliferation and migration

GAN Rong, YANG Xiao, YANG Yingmei, ZHAO Lingxu, MENG Qinghe.

2014, 29(5):  452-458.  DOI: 10.3969/j.issn.1673-8640.2014.05.006

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Objective To study the proliferation and migration abilities of human breast cancer MCF-7 cell line through knocking down microRNA (miRNA)-221 and miRNA-222 expressions and up-regulating tissue metalloproteinase inhibitor 3 (TIMP3). Methods Antisense-miRNA-221(AS-miRNA-221), antisense-miRNA-222(AS-miRNA-222), antisense-miRNA-221/222(AS-miRNA-221/222) and scramble were designed based on Homo sapiens (hsa)-miRNA-221 and hsa-miRNA-222 oligonucleotide sequence and nonsense sequence in miRBase. Lipofectamine^TM2000 was used to transfect AS-miRNA-221, AS-miRNA-222, AS-miRNA-221/222 and scramble into MCF-7 cell line . They were classified into control groups with stable expression [untransfected normal cell group(Control group) and scramble group(Scramble group)] and down-regulated miRNA-221 and miRNA-222 groups(AS-miRNA-221 group, AS-miRNA-222 group and AS-miRNA-221/222 group), respectively, according to G418 screening. After transfecting for 24 h, transfection efficiency was determined by fluorescence microscopy. Real-time fluorescence quantitation polymerase chain reaction (PCR) was used to determine the expressions of miRNA-221 and miRNA-222. The neo gene expression on the plasmid vector was detected in each cell line by reverse transcription PCR. The TIMP3 mRNA level was assessed by real-time fluorescence quantitation PCR, and the expression difference of metalloproteinase 17(ADAM17) protein was determined by Western blotting. The growth curves to describe the low-expressed miRNA-221 and miRNA-222 cells′growth were evaluated by CCK-8. The wound healing model was used to represent the migration ability. Results After transfecting for 24 h, there was an increasing expression of green fluorescence, and the transfection efficiency was high. The expressions of neo gene in Control group were lower than those in Scramble, AS-miRNA-221, AS-miRNA-222 and AS-miRNA-221/222 groups. Compared to Scramble group, the expressions of miRNA-221 and miRNA-222 decreased in AS-miRNA-221, AS-miRNA-222 and AS-miRNA-221/222 groups, and the establishment of AS-miRNA-221/222 down-regulated expression-stable cell line had succeeded. Compared with Scramble group, the expressions of TIMP3 mRNA of AS-miRNA-221 group, AS-miRNA-222 and AS-miRNA-221/222 groups increased, and the level of ADAM17 decreased. The growth curves showed that the proliferation of down-regulated miRNA-221 and miRNA-222 groups had been inhibited. The wound healing model showed that the migration of down-regulated miRNA-221 and miRNA-222 groups had been decreased. Conclusions Through up-regulatingp TIMP3, knocking down miRNA-221 and miRNA-222 can inhibit the proliferation and migration of human breast cancer MCF-7 cell line.

The application significance of TALEN technique for genome research

YING Chunmei, CHEN Yisheng, ZHEN Bing.

2014, 29(5):  459-463.  DOI: 10.3969/j.issn.1673-8640.2014.05.002

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Gene knockout is a broadly applicable approach for efficiently editing any gene of cells and organisms. The transcription activator-like effector nuclease (TALEN) technique is a newly-developed method, and it can target essentially any sequence by comprising a nonspecific DNA-cleaving nuclease fused to a DNA-binding domain, which can be engineered easily. TALEN technique with the capability to quickly and efficiently alter genes have profound impacts on biological research and get potential therapeutic strategies for genetic diseases.

Primary survey for the reference intervals of serum 25-hydroxy vitamin D from adults in Hainan area

XIONG Yu, QIAN Shiyun, XIONG Xiaoquan, CUI Kaimei, CAI Qunfang, WU Qiang.

2014, 29(5):  464-467.  DOI: 10.3969/j.issn.1673-8640.2014.05.008

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Objective To survey the reference intervals of serum 25-hydroxy vitamin D(25OH-D) from adults in Hainan area, and provide scientific reference for reasonable supplement of vitamin D. Methods Basic data of 1 978 healthy subjects(1 008 males and 970 females) with similar livelihood background and different ages were collected, and the level of serum 25OH-D was analyzed by electro-chemiluminescence. The subjects were classified according to sex and age, and the results were analyzed statistically. Results The accuracy and precision of the 25OH-D detection system were good, and the levels of serum 25OH-D had non-Gaussian distribution. The reference intervals were established by percentile (P_2.5-P_97.5). There were statistical significances of 25 OH-D between males and females (P<0.01). There was statistical significance between ≤50 years old and ＞50 years old among males and females(P<0.01). The reference interval of males ≤50 years old was 6.77-26.80 ng/mL, and that of males >50 years old was 4.51-19.73 ng/mL. The reference interval of females ≤50 years old was 5.96-24.79 ng/mL and that of females >50 years old was 3.71-16.34 ng/mL. Conclusions The reference interval for specific sex and age should be established for the population of Hainan area.

Changes and significance of plasma fibrinogen γ levels in preeclampsia pregnant women

ZHU Yuli, LI Qian, CHEN Xiong, LU Shuaijun, LONG Anxiong, TAN Longyi.

2014, 29(5):  468-471.  DOI: 10.3969/j.issn.1673-8640.2014.05.009

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Objective To investigate the levels of fibrinogen γ and its products in plasma of preeclampsia pregnant women, and to study the predictive and aided diagnosis significance in preeclampsia. Methods The clinical data of human chorionic gonadotropin(HCG)-positive pregnant women were followed and collected until delivery, and the plasma specimens were collected. The levels of fibrinogen γ were measured by Western blotting in 31 patients with preeclampsia (15 patients with mild preeclampsia and 16 patients with severe preeclampsia) and 31 healthy controls being enrolled randomly. Results The levels of fibrinogen γ had no statistical significance in plasma of healthy controls during pregnancy(P>0.05). The levels of fibrinogen γ in plasma of patients on 2-7 weeks before the onset of preeclampsia began to rise, and it rose more when preeclampsia happened(P<0.05). Three fibrinogen γ fragments, 25 000, 27 000 and 23 000, appeared in plasma of 1 patient on 2 weeks before preeclampsia happened and 3 patients when preeclampsia happened in 16 patients who developed severe preeclampsia.These fragments appeared in only 1 patient in 15 patients who developed mild preeclampsia. These fragments did not appear in 31 healthy controls. Conclusions Patients before and after the onset of preeclampsia have a synthesis and degradation fibrinogen γ disorder. The synthesis of fibrinogen γ increases, and fibrinogen γ fragments, 25 000, 27 000 and 23 000, appear when fibrinogen γ degradated in some patients. The levels of fibrinogen γ and these fragments may have some significance for predicting and diagnosing preeclampsia.

Study on the correlationship of serum VCAM-1, FGF 2 and other inflammatory cytokines with refractory diabetic foot

ZHANG Ying, WANG Weiling, YANG Qintong, ZHAO Jingjing, ZHOU Lixia, XING Jiayi, ZHAO Yanrong.

2014, 29(5):  472-476.  DOI: 10.3969/j.issn.1673-8640.2014.05.010

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Objective To investigate the relationship of vascular cell adhesion molecule-1 (VCAM-1), fibroblast growth factor 2 (FGF2) and other inflammatory cytokines with the occurrence and development of refractory diabetic foot(DF). Methods A total of 145 patients with refractory DF (DF group) and 65 patients with type 2 diabetes mellitus(T2DM) and non-foot ulcers (T2DM group) were enrolled for the basic clinical data, and fasting blood were collected for the determinations of VCAM-1, FGF2, tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), fasting blood glucose (FBG), 2 h postprandial blood glucose (2 hPG), glycosylated hemoglobin A_1c(HbA_1c), glycated albumin (GA), C-reactive protein (CRP), fibrinogen (FIB), white blood cell count (WBC) and neutrophil ratio (Neu%). The results were analyzed statistically. According to Wagner grade, DF group was classified into Wagner Ⅲ(37 cases), Wagner Ⅳ(92 cases) and Wagner Ⅴ(16 cases). Results Between DF and T2DM groups, sex, body mass index (BMI), FBG, 2 hPG, HbA_1c and GA had no statistical significance (P> 0.05). The levels of VCAM-1, FGF2, TNF-α, IL-6, WBC, Neu%, FIB, CRP, age, duration of diabetes and heart rate in DF group were significantly higher than those in T2DM group(P<0.01).TNF-α was significantly correlated with VCAM-1(r=0.284, P=0.000).Univariate Logistic regression analysis showed that VCAM-1, FGF2, TNF-α, IL-6, age, duration of diabetes, heart rate, WBC, Neu%, FIB and CRP were risk factors for DF occurrence. Multivariate Logistic regression analysis showed that only VCAM-1, TNF-α, FIB and Neu% were the independent risk factors of DF occurrence. The age of Wagner Ⅴ group was significantly lower than those of Wagner Ⅲ and Ⅳ groups(P<0.01), and serum IL-6, CRP, FIB, WBC and Neu% were significantly higher than those of Wagner Ⅲ and Ⅳ groups(P<0.05, P<0.01 ). The levels of FGF2 and HbA_1c in Wagner Ⅴ group were significantly higher than those in Wagner Ⅲ group(P<0.05). Conclusions The elevated levels of serum VCAM-1, FGF2 and other inflammatory cytokines play important roles in the occurrence and development of DF, and we should pay more attention to anti-inflammatory therapy when treating DF.

Clinical significance of Th1, Th2 and Th17 cell determinations among different active stage ankylosing spondylitis patients

ZHONG Naifeng, MA Li.

2014, 29(5):  477-482.  DOI: 10.3969/j.issn.1673-8640.2014.05.011

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Objective To investigate the roles and clinical significance of T helper cell 1(Th1), T helper cell 2(Th2) and T helper cell 17(Th17) in the occurrence and development of ankylosing spondylitis (AS). Methods A total of 78 AS patients(44 cases of low-activity group and 34 cases of high-activity group) and 30 healthy subjects(healthy control group) were enrolled, and their Th1, Th2 and Th17 percentages were determined by flow cytometry(FCM) and intracellular cytokine staining(ICS) technique. The relationship between the percentages of Th1, Th2 and Th17 and the activity of AS was analyzed. Results The percentages of Th1 in AS group, low-activity group and high-activity group were 15.86%±3.30%, 14.18%±2.35% and 17.75%±3.14%, which were significantly higher than that in healthy control group(12.05%±2.35%, P<0.01). The percentage of Th1 in high-activity group was significantly higher than that in low-activity group(P<0.01).The percentages of Th2 in AS group and high-activity group were 1.51%±0.51% and 1.31%±0.41%, which were significantly lower than those in low-activity group (1.90%±0.51%) and healthy control group (1.98%±0.60%, P<0.05).The percentages of Th17 in AS group, low-activity group and high-activity group were 1.57%±0.78%, 1.26%±0.57% and 2.00%±0.86%, which were significantly higher than that in healthy control group (0.82%±0.35%, P<0.05). The percentage of Th17 in low-activity group was significantly lower than that in high-activity group(P<0.01). The relationship between Bath AS disease activity index (BASDAI) and the percentages of Th1 and Th17 showed that there was a positive correlation [correlation coefficient (r) 0.809 and 0.409, P<0.01], and BASDAI with the percentage of Th2 had negative correlation (r= -0.340, P<0.01). Conclusions AS patients′ CD4⁺ T lymphocyte subsets are functionally defective, consequent to the imbalance of cytokine network and immune functional defection, being involved in occurrence and development. Th1, Th2 and Th17 can be as new detection indices for the diagnosis of AS, which could serve as indicators of prognosis.

Establishment and assessment of the cut-off value for ALT and HBV DNA combination determination to diagnose HBeAg negative chronic active hepatitis B

LIU Weiping, YI Qin, YIN Minggang.

2014, 29(5):  483-487.  DOI: 10.3969/j.issn.1673-8640.2014.05.012

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Objective To establish and assess the cut-off value for alanine aminotransferase(ALT), hepatitis B virus (HBV) DNA and aspartate aminotransferase(AST) to diagnose hepatitis B e antigen (HBeAg) negative chronic active hepatitis B. Methods A total of 290 HBeAg negative chronic hepatitis B patients were enrolled and classified into 2 groups: active (213 cases) and inactive(77 cases) groups according to liver biopsy. The diagnosis significance for ALT, AST and HBV DNA load in HBeAg negative chronic active hepatitis B was evaluated by receiver operating characteristic (ROC) curves. Results The areas under ROC curve (AUC) for serum ALT, AST and HBV DNA load were 0.849, 0.807 and 0.867, respectively. For the cut-off values of 30 IU/L for male ALT and 19 IU/L for female ALT and HBV DNA load 1×10⁵ copies/mL, the sensitivity was 90.6%, the specificity was 98.7%, the positive predictive value was 99.5%, and the negative predictive value was 79.2%. The sensitivity of 77.0%, specificity of 63.3%, positive predictive value of 85.4% and negative predictive value of 50.0% were also observed for AST 40 IU/L as the cut-off value. Conclusions The 30 IU/L for male ALT and 19 IU/L for female ALT and HBV DNA load 1×10⁵ copies/mL are suggested to be the cut-off values for discriminating HBeAg negative chronic active hepatitis B.

Serum levels of HE4 and SCCAg and their relationships with biological behavior of endometrial carcinoma

LAO Ming, WU Yun, ZHU Bo, HUANG Wencheng, HUANG Lingsha, WANG Ying, LI Meiqin, ZHU Chunyan, LIU Jinfeng.

2014, 29(5):  488-491.  DOI: 10.3969/j.issn.1673-8640.2014.05.013

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Objective To investigate human epididymal secretory protein 4 (HE4) and squamous cell carcinoma antigen (SCCAg) levels in endometrial carcinoma and their relationships with biological behavior of endometrial carcinoma. Methods Serum levels of HE4 and SCCAg were detected by enzyme-linked immunosorbent assay in 85 patients with endometrial carcinoma, 30 patients with endometrial atypical hyperplasia and 30 healthy women (control group). The clinical signicance of HE4 and SCCAg were analyzed comparatively. Results Serum levels of HE4 and SCCAg were (199.6 ± 31.2) pmol/L and 0.98(0.15-4.85) μg/L in patients with endometrial carcinoma, which were higher than those in patients with endometrial atypical hyperplasia [(91.6 ± 16.8) pmol/L and 0.18(0.02-1.41) μg/L, P<0.05] and control group [(82.5 ± 9.7) pmol/L and 0.13(0.03-1.32) μg/L, P<0.05]. The diagnosis sensitivities of HE4 and SCCAg for the diagnosis of endometrial carcinoma were 63.5% and 14.1%, and the specificities were 96.7% and 100.0%. Serum HE4 and SCCAg levels and the positive rates had increasing trend with stage rising, and the positive rates in advanced stages of endometrial carcinoma (HE4: 90.0% for stage Ⅲ and 100.0% for stage Ⅳ, SCCAg: 30.0% for stage Ⅲ and 25.0% for stage Ⅳ), which were higher than those in early stage(61.1% and 5.6% for stage Ⅰ, P<0.05). For different clinical pathological factors, the positive rate of HE4 in non-endometrial adenocarcinoma was up to 92.0%, which was higher than that in endometrial adenocarcinoma (51.7%, P<0.05). HE4 expression in endometrial carcinoma had the relationship with tumor size, pathological type, depth of tumor invasion and tumor metastasis (P<0.05), and SCCAg expression in endometrial carcinoma had the relationship with tumor metastasis only (P<0.01). Conclusions Serum HE4 has clinical early diagnosis and identification significance for endometrial carcinoma, and it can be used as an indicator for monitoring endometrial carcinoma. However, SCCAg had a little diagnosis significance for the diagnosis of endometrial carcinoma. It only has a certain significance in the course of monitoring and tumor metastasis identification.

Clinical significance of monitoring BK virus in transplanting patients

WU Beiying, CAI Gang, LIN Jiafei, FAN Zhenjia, FAN Qishi.

2014, 29(5):  493-498.  DOI: 10.3969/j.issn.1673-8640.2014.05.015

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Objective To monitor blood and urine BK virus (BKV) load and discuss the influence of sex, age, human cytomegalovirus(HCMV) and immunosuppressor on BKV load and the influence of BKV load on diseases. Methods BKV loads of plasma and urine from patients were measured by real-time fluorescence quantitation polymerase chain reaction(PCR). The influence of plasma HCMV concentration and immunosuppressor concentrantion on BKV load and the influence of high or low BKV load on diseases were analyzed. Results BKV was detected in 242 (12.1%) cases of 1 985 patients with or without transplantation. A total of 211 cases were positive in plasma (10.6%), while 118 cases were positive in urine (5.9%). Bone marrow transplantation, kidney transplantation and none transplantation patients had different BKV positive rates with statistical significance (P<0.01). Almost all cases of BKV positive occurred in the first 3 months after transplantation. There was no statistical significance between HCMV infection and BKV load (P=0.272 9). Among bone marrow transplantation and kidney transplantation patients, the differences of BKV load to plasma immunosuppessor concentration and cyclosporine A(CsA) concentration (>150 ng/mL) had statistical significance (P<0.01), those to tacrolimus (FK506)(>8.0 mg/mL) had no statistical significance (P=0.278 5). For BKV positive and negative patients, there was statistical significance when monitoring serum creatinine (CREA)(P=0.020 7). Conclusions There is no relationship of sex, age and concurrent infection of HCMV infection with BKV replication. However, the concentration of plasma immunosuppressor is related with BKV replication. The secondary damage can be reduced by adjusting the dose of immunosuppressor from continuously monitoring BKV load.

Analysis on the contamination of MGIT liquid culture manual method for Mycobacterium tuberculosis by decontamination

WU Xuebing, CHEN Biying, JIANG Yuan, LI Zhilan, LU Bin, KAN Lixin.

2014, 29(5):  498-500.  DOI: 10.3969/j.issn.1673-8640.2014.05.016

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Objective To analyze the contamination and treatment effect of Mycobacterium grow indicate tube (MGIT) liquid culture manual method for the detection of Mycobacterium tuberculosis. Methods A total of 1 088 sputum specimens from patients suspected pulmonary tuberculosis diagnosed preliminary were collected, and all specimens were respectively determined by MGIT method, improved L-J medium base (L-J) method and sputum smear acid-fast staining, and the contamination rates of MGIT method for the first time and after decontamination were compared with those of L-J method. Results In MGIT method for the first time, the contamination rate of smear positive group was 11.24%, that of smear negative group was 7.29%, and the total contamination rate was 7.90%. In L-J method, the contamination rate of smear positive group was 2.96%, that of smear negative group was 3.80%, and the total contamination rate was 3.68%. The corresponding differences were statistically significant (P<0.05). The contamination rate of smear positive group after decontamination was lower than that of smear negative group, there was statistical significance (P<0.05). Conclusions The MGIT method can be used to decontaminate in contaminated culture and reduce the contamination rate, especially smear negative specimens should be adjusted and increased the concentration of digestion time or sterilized agent before pretreatment, in order to control the first time MGIT method′s contamination rate, reduce the number of decontamination, and recommend that clinicians should control the other bacterial infection before leaving specimens for tuberculosis culture.

Application research on virus load and optimal threshold for the detection of high risk HPV DNA in cervical precancerous lesion

NI Yinghua, SHI Youhao.

2014, 29(5):  501-504.  DOI: 10.3969/j.issn.1673-8640.2014.05.017

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Objective To investigate the application of virus load and optimal threshold for the detection of high risk human papillomavirus(HPV) DNA in cervical precancerous lesion. Methods A total of 805 patients without cervical cancer were performed HPV DNA detection and pathological diagnosis through colposcope biopsy. According to the pathological results, the patients were classified into 86 cases without lesion, 492 cases of chronic cervicitis, 117 cases of low-grade squamous intraepithelial lesion (LSIL)and 110 cases of high-grade squamous intraepithelial lesion (HSIL). The HPV DNA positive rate in each group was compared, and the receiver operating characteristic (ROC) curve was used to determine the optimal RLU/CO value of HPV DNA in cervical squamous intraepithelial lesion. Results The positive rates of HPV DNA detection in no lesion group, chronic cervicitis group, LSIL group and HSIL group were 20.93%, 34.96%, 75.21% and 95.45%, and the virus loads (means) of high risk HPV determination results were 0.355 0, 0.455 0, 47.41 and 286.68. The testing results of cervical squamous intraepithelial lesion were ordered according to the virus loads from small to big, and the abnormal detection rates were 8.29%, 24.42%, 48.94%, 59.66% and 68.54%. According to the ROC curve, the optimal RLU/CO value of cervical squamous intraepithelial lesion was 3.155 0, the sensitivity of the point was 82.2%, while the specificity was 73.9%, and the Youden index was 1.561. The optimal RLU/CO value of HSIL was 5.965 0, the sensitivity of the point was 92.0%, while the specificity was 68.8%, and the Youden index was 1.608. Conclusions The virus load of high risk HPV DNA is directly proportional to the positive rate of cervical lesion detection. Increasing the optimal RLU/CO value properly could improve the specificity and decrease the false positivity of cervical squamous intraepithelial lesion.

Evaluation and comparison on the performance of detecting HbA_1c by TINIA method and IE-HPLC method in HbE patients

WEN Dongmei, ZHANG Xiuming, WANG Weijia, CHEN Yaqiong, SUO Minghuan, XU Quanzhong, WU Jianyang, LI Man, XIAO Jinli, KAN Lijuan, ZHOU Jiashi.

2014, 29(5):  505-512.  DOI: 10.3969/j.issn.1673-8640.2014.05.018

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Objective To evaluate the analysis performance of glycosylated hemoglobin A_1c(HbA_1c) detection by turbidimetric inhibition immunoassay (TINIA), to compare and evaluate the bias with ion exchange high performance liquid chromatography (IE-HPLC) for detecting HbA_1c, and to evaluate the influence of hemoglobin (Hb) E (HbE) on TINIA and IE-HPLC. Methods According to the methodology evaluation document (EP document) and relevant literatures from the Clinical and Laboratory Standards Institute (CLSI), the precision, accuracy, detection range, analysis interference and biological reference interval analysis of HbA_1c detection by TINIA were evaluated and verified. The comparison analysis and bias evaluation of TINIA and IE-HPLC for blood samples with normal Hb structure and blood samples in HbE patients were performed. The correlation with average blood glucose was analyzed. Results The within-run precision by TINIA was < 2.08%, the total imprecision was <2.94%, and the theoretical value with detection mean value was linearly correlated in the concentration range of 4.4%-18.3%(r=0.9994). When Hb, free bilirubin(FBiL), conjugated bilirubin(CBiL) and chyle(CH) concentrations were 9.9-49.6 μmol/L, 65.6-328.0 μmol/L, 65.6-328.0 μmol/L and 3 000-12 000 FTU, the difference percentage was <5%. The manufacturers provided reference interval (4.8%-5.9%) being suitable for clinical laboratories. For the blood samples with normal Hb structure, there was no statistical significance for HbA_1c detection between the 2 methods (P>0.05), and the results of HbA_1c detection by TINIA and IE-HPLC had good correlation (r²=0.999 2). For the blood samples with HbE concentration in the range of 5.4%-54.7%, the average bias by TINIA was -4.1%-1.9%, which was <5%. The interference was almost not affected by HbE, and the correlation of HbA_1c detection results with average blood glucose was good (r=0.998 8). While HbE >6.4%, the average bias of the detection value and the theoretical value for HbA_1c by IE-HPLC was 5.7%-278.7%, the difference percentage was >5%, and the expected theoretical results with the detection value had significant differences, and there was statistical significance between the 2 methods (P< 0.01). Conclusions The performance of HbA_1c detection by TINIA meets the performance requirements of clinical determination, and has a good correlation with that by IE-HPLC without interference by HbE. When HbE>6.4%, IE-HPLC for the detection of HbA_1c has interference, and there will be false increasing.

Assessment of enzymatic method for glycated albumin and its correlation research with the diagnosis of diabetes mellitus

HUANG Weigang, HUANG Sheng, SHEN Jun, LU Daru, WANG Hualiang.

2014, 29(5):  513-517.  DOI: 10.3969/j.issn.1673-8640.2014.05.019

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Objective To evaluate the methodology and performance of serum glycated albumin(GA) determination by enzymatic method as routine method and the correlation of glycosylated hemoglobin A_1c (HbA_1c) with GA, in order to provide a better method for the early diagnosis and effect monitoring of diabetes mellitus. Methods The enzymatic method for GA was evaluated for precision, linearity, bias, carryover rate and drift according to the Clinical and Laboratory Standards Institute (CLSI) EP10-A3. The GA and HbA_1c were determined in 336 patients with diabetes mellitus. The correlation between GA and HbA_1c was analyzed. According to American Diabetes Association (ADA) standard (HbA_1c<7% as blood glucose controlling good, HbA_1c 7%-8% as blood glucose controlling common, HbA_1c>8% as blood glucose controlling poor), the cut-off value of GA/HbA_1c ratio was evaluated by receiver operating characteristic (ROC) curve. Results The total mean imprecision was 1.62%, and the mean bias was 7.58%.The intercept, slope, nonlinearity, carryover rate and drift of enzymatic method were acceptable. The nonlinearity parameter had forward bias when determining high-value, which should be observed further, however, there was no influence on its clinical diagnosis. The correlation analysis indicated that GA was well correlated with HbA_1c[correlation coefficient (r)=0.879, P<0.01]. The optimal cut-off value of GA/HbA_1c ratio for blood glucose controlling was 2.60. The sensitivity was 86.8%, and the specificity was 70.1%. GA/HbA_1c ratio>2.60 was considered as blood glucose controlling poor. Conclusions The routine determination of GA by enzymatic method is available. GA can be used into the diagnosis and effect evaluation of GA. GA/HbA_1c ratio is valuable to monitor blood glucose controlling.

Evaluation on the performance of Reflotron-Hemoglobin dry-chemistry system

CHEN Liting, WANG Jianbiao, LIN Xiaoyi, SHI Hourong.

2014, 29(5):  518-520.  DOI: 10.3969/j.issn.1673-8640.2014.05.020

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Objective To evaluate Reflotron-Hemoglobin dry-chemistry system for the measurement of hemoglobin. Methods The within-run and between-run precisions, linearity, correlation and reference range of Reflotron- Hemoglobin dry-chemistry system was verified. Results The within-run precision was 2.54%-2.84%, and the between-run precision was 0.68%-3.38% for Reflotron-Hemoglobin dry-chemistry system, respectively. The linearity range was 52-191 g/L . The results of Reflotron-Hemoglobin dry-chemistry system were correlated well with those of Beckman-Coulter LH750 system, and the correlation equation wan Y=1.031 6X-2.796 9. The verification coincidence rate for reference range was 100%. Conclusions The within-run and between-run precisions of Reflotron-Hemoglobin dry-chemistry system for the determination of hemoglobin are good with accurate results for both normal and abnormal samples. The reference range can be used in this system.

Application performance evaluation of PENTRA MS60 hematology analyzer

XUE Bingrong, XU Peng, ZHOU Yulian, TIAN Xiaojun, YAN Xinyu, SU Jianrong, YE Ping.

2014, 29(5):  521-527.  DOI: 10.3969/j.issn.1673-8640.2014.05.021

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Objective To evaluate the performance of PENTRA MS60 hematology analyzer(PMS60) for the determination of complete blood count(CBC) and white blood cell (WBC) differential count. Methods The background count, carryover rate(CR), precisions [within-run and inter-day coefficients of variation (CV)], linear range and accuracy of PMS60 were evaluated by fresh blood samples and instrument quality control materials. The bias and correlation of PMS60 and SYSMEX XS800i hematology analyzer(XS800i) were calculated. The microscopy was as a ″gold standard″ of WBC differential count, and the accuracy of WBC differential count was calculated according to the ″gold standard″. Results The background counts of WBC, red blood cell(RBC), hemoglobin(Hb) and platelet(PLT) were all 0. The CR of WBC, RBC, Hb and PLT were 0.09%, 0.33%, 0.54% and 0.10%. The within-run CV of RBC, Hb, hematocrit(HCT), erythrocyte mean corpuscular volume(MCV), mean corpuscular hemoglobin(MCH) and mean corpuscular hemoglobin concentration(MCHC)(normal sample and medical decision level sample) were <1.5%, the within-run CV of WBC were 0.81% and 3.47%, and the within-run CV of PLT were 3.74% and 7.44%. The inter-day CV of RBC, Hb, HCT, MCV, MCH and MCHC(low, middle and high levels) were <2%, the inter-day CV of WBC were 3.47%, 2.99% and 2.49%, and the inter-day CV of PLT were 5.28%, 4.00% and 3.20%. Within the concentration range of clinical samples, the correlation coefficients(r) between the measured values and theoretical values of WBC, RBC, Hb and PLT were >0.998(P<0.01). The slope of linear regression equation was in the range of 1.00±0.05. The relative biases of Hb, HCT, MCV, MCH and MCHC were <3%, and the relative deviation of WBC, RBC and PLT were <6.5%, <3.5% and <9.5%, respectively. The coincidence rate of the relative deviation for CBC was >85%(r＞0.8, P<0.01). Compare to microscopy, the accuracy rates of WBC differential count about lymphocytes, monocytes, granulocytes, eosinophils and basophils were 95%, 95%, 85%, 80% and 75%, respectively. Conclusions The PMS60 has a good performance. It has the advantages of low carryover rate, high precision, wide linear range and reliable accuracy of the results.

Research on 16S rRNA methylase in aminoglycoside-resistant Enterobacteriaceae

WU Qiong, HAN Lizhong, SUN Jingyong, NI Yuxing, CHEN Min

2014, 29(5):  528-534.  DOI: 10.3969/j.issn.1673-8640.2014.05.022

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Objective To investigate the molecular epidemiological characterization and the drug resistance and prevalence mechanism of 16S rRNA methylase in aminoglycoside-resistant Enterobacteriaceae isolated clinically. Methods Gentamicin-and or amikacin-resistant Enterobacteriaceae were screened by disc diffusion method. 16S rRNA methylase genes, aminoglycoside modification enzyme genes and beta-lactamase genes were amplified by polymerase chain reaction(PCR). The conjugal transfer of aminoglycoside-resistant determination was performed. Pulsed-field gel electrophoresis (PFGE) was carried out to analyze genotyping. Results A total of 16 armA gene and 22 rmtB gene 16S rRNA methylase positive isolates were identified. Plasmid conjugation experiments were successful with 30 armA- or rmtB-positive isolates. bla_CTX-M-14, bla_TEM-1 and bla_SHV-12 co-transferred with armA or rmtB were transferred to 11, 20 and 7 isolates. Klebsiella pneumoniae, Escherichia coli and Enterobacter cloacae were divided into 4, 21 and 1 PFGE patterns. Conclusions armA and rmtB are the main types of 16S rRNA methylase in Enterobacteriaceae isolated in our hospital, and rmtB is more prevalent than armA. This 16S rRNA methylase could mediate high-level resistance to aminoglycoside, and the encoding genes are located on plasmid that could transfer among different species. Beta-lactamase genes and fluoroquinolone resistance could be cotransferred with armA or rmtB gene.

The anti-inflammatory role and mechanism of andrographolide on Pseudomonas aeruginosa-induced lung inflammation in rat model

LI Liyan, LI Hongchun, MA Ping.

2014, 29(5):  535-539.  DOI: 10.3969/j.issn.1673-8640.2014.05.023

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Objective To investigate the anti-inflammatory role and mechanism of andrographolide on Pseudomonas aeruginosa (PA)-induced lung inflammation in rats. Methods The rat′s model of PA-induced lung inflammation was established and classified into different time groups(0, 24, 48, 72 and 96h). The white blood cell count and leukocyte classification were determined in blood and bronchoalveolar lavage fluid. Reverse transcription polymerase chain reaction (PCR) was used to determine the level of Toll-like receptor 4 (TLR4) in lung tissue, a peak time point was selected, and the subjects were classified into infection group, solvent group and treatment group(5, 10, 15 and 20μg/g body weight for andrographolide), and the rat blood, bronchoalveolar lavage fluid and lung tissue at the peak time point were used for testing various indicators. Results After PA inflammation, the white blood cell count decreased in 24 h, then increased and reached the peak point at 48 h . The total cell count in bronchoalveolar lavage fluid reached the peak point at 48 h. The mRNA expressions of TLR4 in white blood cell, bronchoalveolar lavage fluid and lung tissue were higher than the others. After receiving andrographolide, the white blood cell count, the total cell count of bronchoalveolar lavage fluid and TLR4 mRNA decreased to various degrees, and had dose-dependent manner. Conclusions PA-induced lung inflammation is related with TLR4 mRNA expression changes, and andrographolide can effectively suppress lung inflammation induced by PA. Its anti-inflammatory mechanism may be achieved through the regulation of TLR4 mRNA expression.

Research on the function of miRNA-195 on bladder cancer cell

CHEN Jing, SUN Fenyong, MA Ji.

2014, 29(5):  540-544.  DOI: 10.3969/j.issn.1673-8640.2014.05.024

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Objective To investigate the influence of microRNA (miRNA)-195 on the proliferation, migration and invasion of human bladder cancer cell 5637. Methods Human bladder cancer cell 5637 was transfected with miRNA-195 by cationic liposomes as miRNA-195 group and transfected with the vector as control group. The expression of miRNA-195 was determined by real-time fluorescence quantitation polymerase chain reaction (PCR). Methyl thiazolyl tetrazolium(MTT) assay, cell counting assay and colony formation assay were used to detect the cell proliferation . Cell migration and invasion were determined by wound healling assay, Transwell assay and Matrigel tumour invasion assay. Results Real-time fluorescence quantitation PCR showed that there was high expression of miRNA-195 after transfection of cell 5637. MTT assay, cell counting assay and colony formation assay demonstrated that miRNA-195 can suppress cell 5637 proliferation in vitro, and there were statistical significance between miRNA-195 and control groups(P<0.05).Wound healing assay, Transwell assay and tumour invasion assay showed that miRNA-195 can suppress cell migration and invasion with statistical significance between the 2 groups(P<0.05). Conclusions MiRNA-195 has inhibitory ability on the proliferation, migration and invasion of bladder cancer cell 5637 cell.

Analysis on the quality levels of national tumor markers among different measuring systems

ZHAO Haijian, ZHANG Chuanbao, WANG Jing, MA Rong, ZHANG Jiangtao, WANG Zhiguo.

2014, 29(5):  545-548.  DOI: 10.3969/j.issn.1673-8640.2014.05.025

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Objective To analyze intralaboratory precisions and interlaboratory variations of the measuring system for national tumor marker detection, and to investigate the quality levels among different measuring systems. Methods External quality assessment(EQA) data were collected from the National Center for Clinical Laboratory in the past 3 years. The measuring results were classified into different groups according to the measuring systems, after excluding the data of "mean ± 3s". The coefficients of variation (CV) were calculated respectively. Internal quality control data and corresponding CV from nationwide participanting laboratories in May 2012 were collected by Web-based EQA software system. Acceptable rates were calculated according to Clinical Laboratory Improvement Amendments(CLIA)′ 88 1/3 total allowable error (TEa), 1/4 TEa and the specifications based on biological variation including the minimal, appropriate and optimal imprecisions. Results After classification based on analysis systems, the average CVs of 4 inlet systems were <10%. However, from the groups of radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) showed large CV. More than 70% of laboratories from most systems met 1/3 TEa requirement. More than 85% of laboratories from most systems met appropriate specification based on biological variation. Conclusions The inlet system 2 shows best interlaboratory precision, and inlet system 5 shows best intralaboratory precision. To ensure the reliability of the measuring results, manufacturers and academic institutions should pay more attention to the traceability of tumor markers, meanwhile clinical laboratories should strengthen their quality controls.

Evaluation on the suitability of allowable total error for tumor markers in Shanghai External Quality Assessment Scheme

ZHANG Jian, ZHU Yuqing, XU Chong, LU Yinhua, GE Danhong.

2014, 29(5):  549-552.  DOI: 10.3969/j.issn.1673-8640.2014.05.026

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Objective To analyze the method variation and actual allowable error range for tumor markers in Shanghai External Quality Assessment Scheme, and to compare the difference between international standards and biological variation requirements in order to investigate the suitability of allowable total error (TEa) for tumor markers. Methods Based on the data of Shanghai External Quality Assessment Scheme from 2010 to 2012, 30 samples were analyzed to calculate synthetic coefficients of variation (CV)of different methods for tumor markers, for 6 times. The actual TEa of tumor markers in Shanghai External Quality Assessment Scheme was calculated by synthetic CV and compared with the Rilibak quality control guide in Germany and the Royal College of Pathologists in Australia (RCPA) quality and biological variation requirements. Results In 6 times of Shanghai External Quality Assessment Scheme, the actual TEa of alpha-fetoprotein(AFP), carcinoembryonic antigen(CEA), carbohydrate antigen(CA) 125, CA153, CA19-9 and prostate-specific antigen(PSA) were 22.63%, 22.43%, 19.30%, 21.56%, 27.43% and 19.55%, which were closed to the Rilibak TEa requirements and biology variation TEa desirable requirements, but was superior to the TEa of RCPA requirements. Conclusions In Shanghai, the actual TEa of tumor markers in Shanghai External Quality Assessment Scheme is closed to the requirements of German Rilibak and slightly below the specification of the more strict Australian RCPA TEa, and TEa in Shanghai External Quality Assessment Scheme can meet the current quality level.

Laboratory diagnosis for one human case infected with babesia

XUE Wujin, LI Lingyun, CHEN Jiaqi.

2014, 29(5):  553-555.  DOI: 10.3969/j.issn.1673-8640.2014.05.027

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Objective To review the laboratory data of one human case infected with babesia, in order to gain experience and improve the diagnosis level. Methods The histograms of red blood cell (RBC), platelet (PLT) and white blood cell(WBC) VCS scatterplot were analyzed. The parasite morphology was observed in peripheral blood and bone marrow smears. DNA extracted from peripheral blood of the patients with plasmodium vivax, voles babesia and babesia DNA-specific primers by polymerase chain reaction(PCR). Results RBC histogram peak reduced, and its width increased. PLT histogram had no abnormality. The non-WBC area of WBC VCS scatter plot had particle group. Peripheral blood and bone marrow smears emerged a large number of quasi plasmodium falciparum parasites. The results of PCR product sequencing showed that the sequencing results had 96% and 99% homology with the 18s ribosomal DNA sequence of voles babesia and babesia. Conclusions The patient is infected with voles babesia.

Calcium sensing receptor and regulation of related signal transduction pathways on the secretion of cytokine

WU Qiuyue, SUN Yihua.

2014, 29(5):  564-569.  DOI: 10.3969/j.issn.1673-8640.2014.05.030

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In this article, we summary the calcium sensing receptor(CaSR) structure, signal transduction and physiological function. The regulation mechanism of CaSR on the secretion of cytokines was described through the related signal transduction pathways, and we prospect the relations between them.

Common defects and relative suggestions for English abstract writing of laboratory medicine manuscripts

DONG Yueying.

2014, 29(5):  570-573.  DOI: 10.3969/j.issn.1673-8640.2014.05.031

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Abstract is an important part of one science and technological manuscript, and it can express the main and general idea of one manuscript. With the development of science and technological manuscripts for internet, information and international aspects, as a pathway for the communication with foreign readers and journals, there is a significant role of English abstract for a whole manuscript, so we should pay more attention for English abstract writing. This manuscript here describes the partial common defects and relative suggestions for English abstract writing as Laboratory Medicine for an example. I hope that it could provide some advice and reference for submitting authors to write English abstract.