Table of Content

30 August 2015, Volume 30 Issue 8

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Application of urinary hepcidin-25 determination in the early-stage diagnosis of iron deficient syndrome among children

XU Shuangqing, ZOU Xiaofeng, MA Guoxiang, XU Keqian

2015, 30(8):  777-781.  DOI: 10.3969/j.issn.1673-8640.2015.08.001

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Objective To study the diagnostic performance of urinary hepcidin-25 for the early-stage diagnosis of iron deficient syndrome (IDS) among children. Methods By enzyme-linked immunosorbent assay (ELISA), urinary hepcidin-25 levels were determined in 25 cases of iron depletion(ID) stage, 25 cases of iron deficient erythropoiesis(IDE) stage, 25 cases of iron deficient anemia(IDA) and 25 healthy children (control group). The cut-off values of urinary hepcidin-25 for ID, IDE and IDA and corresponding diagnostic performance were analyzed by receiver operating characteristic(ROC) curve. Results There was statistical significance of urinary hepcidin-25 levels in comparison between any 2 groups of ID, IDE, IDA and control groups (P<0.05). The areas under ROC curves for urinary hepcidin-25 for differentiating ID, IDE and IDA groups from control group were 0.865, 0.974 and 0.998, respectively. The areas under ROC curves for urinary hepcidin-25 for differentiating IDE from IDA, differentiating IDE from ID, differentiating IDE from the mixing group of control with ID were 0.872, 0.870 and 0.922, respectively. Conclusions The determination of urinary hepcidin-25 level is a simple and non-invasive test, and it might have application significance in the early-stage diagnosis of IDS among children.

Response surface method to verify amylase determination procedure's optimum pH, chloride ion and EPS concentrations

MENG Shuting, WANG Huimin, JI Huoyan, WANG Jianxin, XU Lili, YUAN Ruoyu, GU Wenchao.

2015, 30(8):  782-786.  DOI: 10.3969/j.issn.1673-8640.2015.08.002

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Objective To confirm that response surface method (RSM) is a method for amylase (AMY) reference determination procedure with optimum conditions. Methods The single factor analysis of variance was used to study pH, chloride ion (Cl^-) and ethylidene-4-nitrophenyl-alpha-D-maltoheptaoside(EPS) concentrations in order to know whether they had statistical significance for AMY activity. Box-Behnken design was used to study the combined influence of pH (6.9-7.1), Cl^- (50-90 mmol/L) and EPS (4.5-5.5 mmol/L) concentrations on AMY activity. The quantitative relation model on pH, Cl^- and EPS concentrations for AMY activity was established in order to obtain their optimum combination. Results There was statistical significance on the influence of the 3 factors on AMY activity (P<0.05). The one-shot and second-order effects of pH,Cl^- and EPS had statistical significance on AMY activity (P<0.01). Cl^- and EPS interacted significantly (P<0.05).The optimum conditions for AMY were pH 7.02,Cl^- 72.79 mmol/L and EPS 5.07 mmol/L. Conclusions The optimum conditions of pH, Cl^- and EPS have no difference with those recommended from the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC). RSM is a method with optimum conditions to confirm the reference determination procedure.

Significance on the detection of (1,3)-beta-D glucan and CD4⁺T lymphocyte count for the diagnosis of invasive fungal disease in patients with AIDS

LIU Mingbo, LIANG Xin, WENG Guoqing, YAO Qinjiang, HUANG Sulan, WEI Lianghong.

2015, 30(8):  787-790.  DOI: 10.3969/j.issn.1673-8640.2015.08.003

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Objective To investigate the diagnosis significance of the detection of (1,3)-beta-D glucan (BDG) assay(G test) and CD4⁺T lymphocyte count for invasive fungal disease (IFD) in patients with acquired immune deficiency syndrome (AIDS). Methods A total of 297 patients with AIDS from the Tenth Affiliated Hospital of Guangxi Medical University were enrolled from January 2013 to May 2014. They were suspected to suffer from IFD. Before receiving anti-fungal treatment, G test and CD4⁺T lymphocyte count were performed and detected simultaneously. According to the diagnosis standard of IFD, the patients were classified into IFD group (including possible, probable and proven diagnosis) and non-IFD group. G test for diagnosing IFD was analyzed for sensitivity and specificity, respectively. G test and CD4⁺T lymphocyte count were combined to evaluate the diagnosis sensitivity and specificity. Results The sensitivity and specificity of G test for diagnosing IFD in AIDS patients were 75.3% and 87.4%, and the receiver operating characteristic (ROC) curve of G test was analyzed, and the area under the curve was 0.861 (95% confidence interval 0.720-0.865). The sensitivity and specificity were 92.9% and 88.8% in the combined determination of G test and CD4⁺T lymphocyte count. Conclusions G test is useful in the diagnosis of IFD among patients with AIDS, and the clinical application significance is improved when it is combined with CD4⁺T lymphocyte count for diagnosing IFD with AIDS.

The diagnosis significance of urinary sediment determination for male gonococcal urethritis

CHEN Kun, XU Yu, XU Jiaxue, ZHU Min, GUAN Ming.

2015, 30(8):  791-793.  DOI: 10.3969/j.issn.1673-8640.2015.08.004

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Objective To investigate the diagnosis significance of urinary leukocyte count and bacterium count for male gonococcal urethritis. Methods A total of 140 urine specimens for male patients with urethritis were determined for urinary leukocyte count and bacterium count by UF-1000i automatic urine analyzer, which the samples were then concentrated and smeared. The specimens of Gram-negative diplococcus were confirmed as the cases of gonococcal urethritis. The evaluation parameters such as sensitivity and specificity were counted statistically to diagnose male gonococcal urethritis. Results The sensitivity was 92.0%, the specificity was 80.0%, the positive predictive value was 71.9%, the negative predictive value was 94.7%, and the area under receiver operating characteristic (ROC) curve was 0.824 for diagnosing gonococcal urethritis, when urinary leukocyte count was ≥300/μL, and bacterium count was ≤300/μL. Conclusions Urinary leukocyte count and bacterium count show significance in the diagnosis of male gonococcal urethritis.

The diagnosis significance of plasma lipopolysaccharide determination for Gram-negative bacterium infection in organ transplantation recipients

TANG Qingqin, HU Zhide, LIU Yaoting, YANG Min, WEI Tingting, MA Ning, QIN Baodong, FU Haitao, ZHONG Renqian.

2015, 30(8):  794-797.  DOI: 10.3969/j.issn.1673-8640.2015.08.005

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Objective To evaluate the diagnosis significance of plasma lipopolysaccharide (LPS) determination for Gram-negative bacterium (GNB) infection in organ transplantation recipients. Methods The clinical data of 160 liver transplantation and kidney transplantation patients were analyzed retrospectively. According to the results of bacterium isolation culturing, there were 2 groups, GNB group and control group. The LPS concentrations were determined by dynamic turbidimetric assay. Receiver operating characteristic (ROC) curve was used to analyze the diagnosis significance of LPS for GNB infection. Results LPS in GNB group was 33.58(22.12, 73.59)pg/mL, while it in control group was 5.00(5.00,9.78)pg/mL. The difference was statistically significant (P<0.05). The area under ROC curve was 0.834 (95% confidence interval 0.764-0.903). The LPS determination sensitivity and specificity were 81.01% and 88.89%, respectively, at the cut-off value of 20 pg/mL. Conclusions LPS is an effective diagnosis biomarker for GNB infection in organ transplantation recipients.

Surveillance analysis on bacterial drug resistance at Tongling in 2014

TANG Jibin, HU Zhijun, ZHANG Sheng, JIAO Ruibao.

2015, 30(8):  798-803.  DOI: 10.3969/j.issn.1673-8640.2015.08.006

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Objective To investigate the antimicrobial susceptibility and drug resistance of clinical isolates at Tongling People's Hospital. Methods A total of 3 912 nonduplicated clinical isolates were collected in 2014. Kirby-Bauer disc diffusion method was employed to study the antimicrobial susceptibility. The data were analyzed by WHONET 5.6 software according to the Clinical and Laboratory Standards Institute (CLIS) 2013 breakpoints. Results Of the clinical isolates, Gram-negative microorganisms, Gram-positive microorganisms and fungi accounted for 74.5%, 24.6% and 0.9%, respectively. The overall prevalence of methicillin-resistant Staphylococcus aureus (MRSA) was 39.0%, and it was 65.1% for methicillin-resistant coagulase negative Staphylococcus (MRCNS). The resistance rates of methicillin-resistant isolates to beta-lactams and other antimicrobial agents were much higher than those of methicillin-susceptible isolates. No staphylococcal isolate was found being resistant to vancomycin or teicoplanin. Enterococcus faecalis showed relatively low resistance to ampicillin, penicillin and nitrofurantoin. Enterococcus faecium were more resistant than Enterococcus faecalis to most antibiotics tested. Approximately 49.1% of Escherich coli and 50.8% of Klebsiella isolates produced extended-spectrum beta-lactamases(ESBLs). The ESBLs-producing isolates were significantly more resistant to most antibiotics than the corresponding non-ESBLs-producing isolates. The prevalence of carbapenem-resistant isolates was 27.5% in Klebsiella and 2.3% in Escherich coli. The percentage of Acinetobacter baumannii isolates resistant to imipenem and meropenem was 76.7% and 77.0%, respectively. The 28.4% and 27.7% of Pseudomonas aeruginosa isolates were resistant to imipenem and meropenem. Nearly 93.6% Pseudomonas aeruginosa isolates were susceptible to amikacin. Conclusions There appears a trend of increasing durg resistance for the clinical isolates in this hospital, especially carbapenem-resistant Enterobacteriaceae, which is of great concern. It is mandatory to take effective antibiotic policy and infection control measures.

Determinations of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in serum by liquid chromatography tandem-mass spectrometry

XU Fengxian, YU Jiaping.

2015, 30(8):  821-824.  DOI: 10.3969/j.issn.1673-8640.2015.08.012

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Objective To establish a method for the determinations of 25-hydroxyvitamin D2[25(OH)D2]and 25- hydroxyvitamin D3[25(OH)D3] contents in serum by liquid chromatography tandem-mass spectrometry (LC-MS/MS). Methods After proteins in serum were precipitated by ethanol, liquid-liquid extraction by n-Hexane, then the residuals were re-dissolved in acetonitrile and analyzed by LC-MS/MS in the positive electrospray ionization(ESI+) mode and multiple reaction monitor(MRM) mode. The quantitative analysis for 25(OH)D2 and 25(OH)D3 contents was carried out by deuterium isotope as internal standard, and the methodology validation was performed. Results The within-run precision of 25(OH)D2 and 25(OH)D3 was 0.88%-7.69%, and between-run precision was 1.56%-9.90%. The good correlation coefficients were 0.999 1 and 0.999 9 at the concentration of 0.5-10.0 ng/mL 25(OH)D2 and 5-100 ng/mL 25(OH)D3, respectively. The accuracy of quality control materials of 25(OH)D2 and 25(OH)D3 was 95.5%-101.2%. 25(OH)D2 content did not have statistical significance(P>0.05), and 25(OH)D3 content had statistical significance(P<0.05)between males and females of 281 volunteers. Conclusions This method is sensitive, accurate and stable, satisfying the quantification requirements of 25(OH)D2 and 25(OH)D3 in serum, and it can be used in clinical analysis.

Determinations of serum 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 concentrations in cord blood by liquid chromatography tandem-mass spectrometry

ZHAO Li, WANG Weiye.

2015, 30(8):  825-829.  DOI: 10.3969/j.issn.1673-8640.2015.08.013

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Objective To establish a method for determining the concentrations of 25-hydroxyvitamin D2[25(OH)D2] and 25-hydroxyvitamin D3[25(OH)D3] in cord blood. Methods The serum samples from cord blood were pretreated by acetonitrile and ZnSO₄.High performance liquid chromatography tandem-mass spectrometry (LC-MS/MS) was used for the determinations of 25(OH)D2 and 25(OH)D3 concentrations. The analysis was carried on a Poroshell^? 120 EC-C18 column. Electrospray ionization(ESI), multiple reaction monitor (MRM), nebulizer 35.0 psi, gas temperature 180℃, sheath gas temperature 350℃ and sheath gas flow 11.0 L/min were performed. The column temperature was 50℃ with linear gradient elution using methanol(80%)and water(20%), and the flow rate was 0.5 mL/min. The quantitative analysis was performed by selective reaction monitoring mode of heater ESI. Results A good linearity was detected in the linear range of 3.125-100 ᠜g/L for 25(OH) D2 and 25(OH) D3 (R²=0.998 7 and 0.999 1). Blood samples had frozen and thawed for 3 times. Both intra-day and inter-day relative standard deviations (RSD) were <15%. The quality control materials of serum 25(OH)D2 and 25(OH)D3 were analyzed, and the results met the requirements of quality control. Conclusions LC-MS/MS is sensitive, rapid and suitable for the determinations of serum 25(OH)D2 and 25(OH)D3 concentrations in cord blood.

Taqman-MGB method for determining CYP3A5 rs776746 site SNP and the influence of this site on the metabolism of tacrolimus

JIN Lilan, CAI Gang, LIN Lin, WANG Xianghui

2015, 30(8):  830-834.  DOI: 10.3969/j.issn.1673-8640.2015.08.014

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Objective To establish Taqman-MGB real-time fluorescence polymerase chain reaction (PCR) for determining CYP3A5 rs776746 site single nucleotide polymorphism (SNP), and to analyze the influence on the metabolism of tacrolimus. Methods The primers and double-labeled probes were designed according to the genotype of CYP3A5 rs776746 site, and the Taqman-MGB real-time fluorescence PCR for SNP was established and evaluated. A total of 170 whole blood DNA samples from patients who took tacrolimus were collected, and CYP3A5 genotype was determined by Taqman-MGB real-time fluorescence PCR. The relationship between CYP3A5 genotype and tacrolimus concentration to dose ratio was analyzed. Results The established Taqman-MGB method can discriminate CYP3A5 rs776746 site efficiently with good reproducibility and specificity and had a detection limit of 0.01 ng for DNA. The results of Taqman-MGB method were consistent with those of sequencing. Among the 170 samples, 18 samples belonged to CYP3A5*1/*1(10.59%), 65 samples were CYP3A5*1/*3(38.24%) and 87 samples were CYP3A5*3/*3(51.18%). Among the samples collected, 104 samples belonged to renal allograft recipients, and the mean tacrolimus concentrations to dose ratios for 3 genotypes were (52.47±35.96), (77.79±33.80) and (134.80±79.35) (ng/mL)/(mg·kg^-1·d^-1), and there was statistical significance (P<0.05). Conclusions The Taqman-MGB method for determining CYP3A5 rs776746 site SNP is established, which has high sensitivity, specificity and reproducibility and is a rapid method for clinical practice. Through primary analysis, the existence of CYP3A5*3 genotype do weaken the ability of metabolizing tacrolimus.

Establishment of an ELISA for the quantitative measuring of glucagon-like peptide 1 and the primary clinical application

SUN Lishan, WANG Mingdong, LU Liu, LIU Yingbing, LU Lin, GUO Jinhu, FAN Lieying

2015, 30(8):  835-840.  DOI: 10.3969/j.issn.1673-8640.2015.08.015

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Objective To establish an enzyme-linked immunosorbent assay (ELISA) for the quantitative measuring of glucagon-like peptide 1(GLP-1) in serum and to investigate the application in the individualized treatment of type 2 diabetes mellitus (T2DM) patients. Methods Using a polyclonal antibody as capture antibody, another monoclonal antibody as labeled antibody, serial dilutions as standard material, an ELISA was established for the quantitative measuring of GLP-1 in serum, and the biological reference interval was also established. All the subjects were classified into 3 groups, normal glucose tolerance (NGT) group (50 cases), impaired glucose tolerance (IGT) group (52 cases) and T2DM group (68 cases). The blood samples were collected at fasting time, 1 h after taking glucose and 2 h after taking glucose. The dynamic change of GLP-1 was evaluated with routine biological items, in order to evaluate the clinical application significance of GLP-1 for the treatment of T2DM. Results The established method had an good quantitative correlation in the range of 23.5-1 490 nmol/L(R²=0.999, P<0.05). The biological reference interval for adults was (79.5±15.6) nmol/L. The GLP-1 in T2DM group was higher than those in NGT and IGT groups (P<0.05). According to the dynamic change of GLP-1 concentration in the oral glucose tolerance test, the deficient secretion of GLP-1 after taking glucose among patients in IGT and T2DM groups could be concluded. Conclusions The ELISA for the quantitative measuring of GLP-1 in serum is successfully established. This method could be used to dynamically monitor the secretion of GLP-1 in IGT and T2DM patients and would be helpful for the individualized treatment of T2DM.

Analysis of trace elements in rabbit kidney with high-adipose diet by ICP-AES

WU Yunkai, WANG Hu, WEI Wenmei, GU Zhihong, WANG Yuan

2015, 30(8):  841-843.  DOI: 10.3969/j.issn.1673-8640.2015.08.016

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Objective To investigate the content changes of Fe, Mn, Cu, Zn, Se, Pb and Cd in rabbit kidney with atherosclerosis (AS). Methods The model of rabbit kidney with AS was reproduced by a high-adipose diet, kidney was obtained, the samples were microwave-digested with miscible liquids of nitric acid and peroxide of hydrogen, and then the Fe, Mn, Cu, Zn, Se, Pb and Cd contents were determined by inductive coupled plasma atomic emission spectrometry (ICP-AES). Results Serum total cholesterol (TC) and Zn, Fe, Se and Cd contents in normal control and high-adipase groups had statistical significance (P<0.05). Cd contents of the 2 groups were significantly higher than that in feed sample (P<0.05). Conclusions Mn, Cu, Zn and Se contents in high-adipose group decline, which may have some relation with the intake of foods and the abnormality of lipoprotein metabolism. Cd accumulates easily in kidney.

Assessment on the validity of biochemical determination results after internal quality control out-of-control

GE Yajuan, XU Qunhuan, ZHANG Qing

2015, 30(8):  844-846.  DOI: 10.3969/j.issn.1673-8640.2015.08.017

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Although both clinical laboratory administrators and ISO 15189 require to assess determination results from the previous batch of patient specimens after appearing internal quality control out-of-control, they do not provide the detail methods to evaluate them. In this article, we mainly investigate to assess the validity of biochemical determination results after appearing internal quality control out-of-control according to 1/2 allowable total error providing by the external quality assessment of the National Center for Clinical Laboratories (NCCL) with considering control materials, the rules of internal quality control, clinical significance and so on. The results of 33 routine biochemical items were analyzed and showed that there were 661 out-of-control cases in 2013. After the assessment, 9 cases required to withdraw the test results in the previous batch of patient specimens, accounting for 1.3% of total out-of-control cases. How to set rational quality control standard and save resources in accordance with the actual situation of the laboratory is worth thinking for clinical laboratory administrators.

The laboratory characteristics of multiple myeloma

WANG Wei.

2015, 30(8):  847-851.  DOI: 10.3969/j.issn.1673-8640.2015.08.018

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Multiple myeloma(MM) is a plasma cell monoclonal malignant proliferative disease. The myeloma cells infiltrate bone and soft tissue, produce M protein and cause bone destruction, anemia, renal insufficiency, repeated infection and so on. Through the statistics, the incidence of MM in all malignant tumors accounts for about 1%, and its incidence in blood system of malignant tumor is more than 10%.The pathogenesis of MM is not fully clear, the complex and various clinical symptoms lead to misdiagnosis or missed diagnosis easily. The difference of laboratory examination may lead to difficult disease diagnosis and differential diagnosis. Therefore, investigating the clinical and laboratory diagnosis characteristics of MM has clinical significance for the early detection, early diagnosis and early treatment of MM. In this paper, the clinical and laboratory diagnosis characteristics of MM are reviewed in detail.

Application of chromatography in the detection of arachidonic acid metabolites

JIANG Liansheng, TAN Longyi

2015, 30(8):  852-856.  DOI: 10.3969/j.issn.1673-8640.2015.08.019

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Arachidonic acid (AA) metabolite is a series of material produced by lipid peroxidation of (AA), and it is involved in many physiological and pathological processes. AA metabolites may be potential biomarkers for some diseases. Determining their quality and quantity accurately is significant in the occurrence, development, diagnosis and prediction of some diseases. Nearly 70 kinds of AA metabolites have been found, and they are all small molecule compounds. Conventional determination technology can't distinguish AA metabolites as their similar molecular structure. The development of chromatography and mass spectrometry provides a reliable technical method for the determination of AA metabolites. This paper gives a general review of the related advances.

The progress of anti-carbamylated protein antibody

CHEN Yiqiong, ZHAO Di, LI Zhi.

2015, 30(8):  857-860.  DOI: 10.3969/j.issn.1673-8640.2015.08.020

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The key to the treatment of rheumatoid arthritis(RA) is early diagnosis and treatment. It becomes urgent to find sensitive and specific biomarkers for the early diagnosis of RA. In recent years, anti-carbamylated protein (Carp) antibody is attention. Anti-Carp antibody can improve the susceptibility of anti-citrullinated protein antibody (ACPA)-negative RA patients, and the anti-Carp antibody presents in about 30% ACPA-negative RA patients. Like ACPA, anti-Carp antibody is also a biomarker for the early diagnosis of RA. This paper will give a review of the progress.

The research progress in respiratory virus laboratory testing technology

YE Xingchen, YANG Haiou, WANG Jing.

2015, 30(8):  861-865.  DOI: 10.3969/j.issn.1673-8640.2015.08.021

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Respiratory virus is one of main factors that threatens human health, so it is significant to identify pathogenic microorganisms accurately and rapidly. This article introduces some traditional or new laboratory testing technologies that have been applied to respiratory virus determination nowdays, including virus isolation culture technique, immunological technique, nucleic acid sequence amplification technique, chip technology, next generation sequencing technique, mass spectrometry (MS) technique and so on. These technologies are applied into clinical diagnosis, curative effect observation, scientific research and investigation of respiratory virus by their characteristics.

Clinical application of serum biomarkers for ovarian cancer

SONG Binbin, PAN Baishen

2015, 30(8):  866-870.  DOI: 10.3969/j.issn.1673-8640.2015.08.022

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Ovarian cancer is the fifth cause of cancer death in women, as well as the cause of deaths in gynecological cancers. Unlike breast cancer, many women die of ovarian cancer because there is no obvious symptom and no screening test in early stages. Nowadays, only a limited number of tumor markers for ovarian cancer has been cleared by U.S. Food and Drug Administration (FDA). In general, tumor marker applications range from diagnosis and curative effect monitoring to predicting prognosis. The article will review the currently available serum markers for ovarian cancer and their clinical application.