Table of Content

30 May 2015, Volume 30 Issue 5

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The application of mass spectrometry in clinical biochemistry

ZHANG Ziqiang, LI Yan

2015, 30(5):  407-409.  DOI: 10.3969/j.issn.1673-8640.2015.05.001

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Recently, mass spectrometry has been playing more and more important roles in clinical laboratory medicine. The development of gas or high-performance liquid chromatography and tandem mass spectrometry technologies has expanded the application of mass spectrometry, and it becomes an efficient tool for clinical biochemical determination. This article introduces the technical principle of mass spectrometry and presents the utilization in clinical biochemistry, such as newborn screening for genetic diseases and so on. Mass spectrometry has shown great potential with the advantages of high specificity, high sensitivity and low limit of determination in clinical biochemistry.

Application of mass spectrometry for the clinical diagnosis of inborn error of metabolism

ZHANG Chunhua

2015, 30(5):  410-415.  DOI: 10.3969/j.issn.1673-8640.2015.05.002

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For the clinical diagnosis of inborn error of metabolism, the technique of mass spectrometry is considered to be an important tool. Gas chromatography/mass spectrometry can be used in the screening and diagnosis of more than 130 inborn errors of metabolism. Tandem mass spectrometery can screen about 30 fatty acid disorders and amino acid disorders, so it is applied into neonatal screening around the world. The number of laboratories using mass spectrometry for the diagnosis of inborn error of metabolism is increasing, but the organization of mass spectrometry analysis is very difficult. Internationalization management is the precondition for the quality control of inborn error of metabolism determination in clinic. The on-line mass spectrometry network system can overcome difficult procedure and keep high quality for laboratory management.

Establishment on a LC-MS/MS and its performance characteristic evaluation for the determination of serum 25-hydroxyvitamin D

SONG Binbin, QIN Jiaqian, PENG Yingfei, ZHANG Chunyan, WU Jiong, WANG Beili, GUO Wei, PAN Baishen

2015, 30(5):  416-420.  DOI: 10.3969/j.issn.1673-8640.2015.05.003

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To establish a liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the quantitation determination of serum 25-hydroxyvitamin D [25(OH)D] and perform the general method performance verification. The LC-MS/MS was used for the quantitation determination of 25(OH)D2 and 25(OH)D3 in serum by Waters^® Xevo^TM TQ MS ACQUITY UPLC^® mass spectrometry instrument. According to US Food and Drug Administration (FDA)'s guideline (Guidance for Industry Bioanalytical Method Validation), the general method performance verification was performed for linearity, determination limit, precision and accuracy. The linear ranges of 25(OH)D2 and 25(OH)D3 were 6.25-500.00 nmol/L. The LC-MS/MS had quantitation determination limits of 2.50 nmol/L for 25(OH)D2 and 1.25 nmol/L for 25(OH)D3. The within-run and between-run coefficients of variation (CV) of 25(OH)D2 and 25(OH)D3 were <4% and <6%, respectively. The recovery rates were 93.26%-112.16%. The result of the Vitamin D External Quality Assessment Scheme (DEQAS) interlaboratory quality assessment had a bias <10%. The basic performance of LC-MS/MS meets the evaluation standards, and LC-MS/MS is sensitive and accurate for detecting the concentrations of 25(OH)D2 and 25(OH)D3 in serum.

Liquid chromatography-tandem mass spectrometry for uric acid and its comparison with clinical routine determination method

ZHANG Haichen, LI Shuijun, SUN Hewei, SONG Yunxiao

2015, 30(5):  422-426.  DOI: 10.3969/j.issn.1673-8640.2015.05.004

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To establish a liquid chromatography-tandem mass spectrometry(LC-MS/MS)for serum uric acid, and to compare LC-MS/MS with clinical routine determination methods. Uric acid-¹⁵N₂ was added as internal standard, serum samples were precipitated with acetonitrile and dried with nitrogen flow. A reversed-phase chromatographic separation was performed on Capcell C18 MGⅢ analytical column by using 5 mmol/L ammonium acetate plus 0.1% acetate acid in water and methanol(90∶10,v/v)as mobile phase. The flow rate was 0.3 mL/min. Uric acid and internal standard were monitored by a negative electrospray ion-tandem mass spectrometry system using ion transitions of 167/124 amu (quantitation) and 167/96 amu (qualitation) for uric acid and 169/125 amu for uric acid-¹⁵N₂. After being validated, the LC-MS/MS was compared with the uricase ultraviolet (UV) method and uricase colorimetric (UC) method. The LC-MS/MS was validated over a concentration range of 30-952 μmol/L. Within-run and between-run precisions were 2.01%-6.23% and 4.55%-8.08%, respectively. The accuracy was 96.5%-103.4%. The retention time of uric acid was 1.5 min, and total run time was 3 min. The linear correlation formulas among LC-MS/MS, uricase UV method and uricase UC method were Y_UV=0.898X_LC-MS/MS +2.15, r=0.978 and Y_UC=0.845X_LC-MS/MS+22.15, r=0.983. The average biases were 1.56%±0.65% between LC-MS/MS and the National Institute of Standards and Technology (NIST) standard reference material and -0.34%-3.05% between LC-MS/MS and correctness verification samples, 2014 from the National Center for Clinical Laboratory. Serum uric acid can be simply and accurately measured by LC-MS/MS. There is good comparability between LC-MS/MS, uricase UV method and uricase UC method.

Research on the determination of serum steroid hormones by isotope dilution HPLC-MS/MS

ZHOU Yafei, WANG Yueting, YU Jiaping

2015, 30(5):  427-432.  DOI: 10.3969/j.issn.1673-8640.2015.05.005

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To establish a rapid, sensitive and stable isotope dilution high performance liquid chromatography-tandem mass spectrometry(ID-HPLC-MS/MS) for simultaneous quantitative determination of dehydroepiandrosterone(DHEA), 17 alpha-hydroxyprogesterone(17α-OHP4), androstenedione(AD), estrone(E1), corticosterone(CORT), dihydrotestosterone(DHT), pregnenolone(P5)and 17-hydroxylpregnenolone(17-OHP5). The steroid hormones were extracted from human serum by methyl tert-butyl ether(MTBE), and the supernatant was treated with hydroxylamine to produce post-column derivatives before detection. Steroid hormones were separated by Phenomenex reversed-phase C18 with precolumn. Water containing 0.1% formic acid and methanol containing 0.1% formic acid were used as mobile phase A and B,respectively. Multiple reaction monitoring(MRM)with the positive ion detection mode was applied to selectively detect these 8 steroid hormones ionized by electrospray ionization (ESI) interface, and the quantitative analysis for them was carried out by using deuterium isotope as internal standard. The lower limits of quantitation(LLOQ)of these 8 hormones can reach 0.03-0.625 ng/mL on the basis of signal-noise ratio (S/N) ≥10. The correlation coefficients(r)were ≥0.998 0 with a good linear at the concentration of 0.05-50.00 ng/mL. The within-run precision was 1.93%-13.81%, and the between-run precision was 4.90%-16.18%. The recoveries were 80.0%-130.6%. The ID-HPLC-MS/MS is established for simultaneous quantitative determination of 8 kinds of steroid hormones. It has the advantages of sensitivity, specificity and accuracy, which is suitable for the separation and determination of steroid hormones for human serum samples within 10 min.

Establishment of a LC-MS/MS for the determination of urinary catecholamines

PENG Yingfei, WU Jiong, GUO Wei, CHEN Fangjun, QIN Jiaqian, XU Wen, PAN Baishen

2015, 30(5):  433-436.  DOI: 10.3969/j.issn.1673-8640.2015.05.006

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To establish a liquid chromatography-tandem mass spectrometry(LC-MS/MS) for the determination of urinary catecholamines, including epinephrine(E), norepinephrine(NE) and dopamine(DA). E, NE and DA were separated by WATERS ACQUITY UPLC^® HSS T3 (2.1 mm×100 mm,1.8 μm column) using E-d3, NE-d6 and DA-d4 as internal standards. The mobile phase consisted of 2‰ formic acid aqueous solution (A1) and methyl alcohol (B1, purity=99.9%). The flow rate of 0.50 mL/min was applied to the chromatographic column. Flow starting was performed with A1∶B1=95∶5. Electrospray ionization (ESI) was operated in positive ion mode. The LC-MS/MS was used, with linearity, accuracy (recovery rate), imprecision and lower limit of determination being evaluated. A total of 65 healthy subjects were enrolled, and the reference ranges for E, NE and DA were established. The retention times of E, NE and DA were 0.58, 0.89, 0.63 min, respectively. The determination range was from 0.50 ng/mL to 800.00 ng/mL. The mean recovery ranges were 97.95%, deter mination range 97.78% and 101.03%. The lower limits of determination for E, NE and DA were 0.25, 2.50 and 2.50 ng/mL. The reference ranges were 5.4-25.8 μg/24 h for E, 28.5-73.1 μg/24 h for NE and 269.1-420.5 μg/24 h for DA. The LC-MS/MS for the simultaneous determination of E, NE and DA has been established and is suitable for clinical application. It can provide reliable information for the diagnosis of phaeochromocytoma.

Liquid chromatography-tandem mass spectrometry for the determination of serum digoxin and its external quality assessment

SUN Hewei, LIU Haiming, LI Shuijun, YU Chen, ZHU Jianmin

2015, 30(5):  437-441.  DOI: 10.3969/j.issn.1673-8640.2015.05.007

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To establish a liquid chromatography-tandem mass spectrometry(LC-MS/MS)for therapeutic drug monitoring(TDM)of serum digoxin. An isotope dilution LC-MS/MS was realized by tert-butyl methyl ether extraction, reversed-phase separation and positive electrospray ionization mass spectrometry detection. The results of external quality assessment(EQA)of digoxin TDM program of the National Center for Clinical Laboratory by LC-MS/MS were analyzed and compared with those of electrochemiluminescence immunoassay. The linear range was 0.05-5.00 ng/mL. The precision was 1.0%-4.3%, and the accuracy was 101.3%-108.9%. The recovery rate was 95.8%-97.5%, and the matrix effect was 95.7%-97.6%. The total run time was 3 min per injection. Good stability was observed on serum undergoing 3 cycles of frozen-thaw, at room temperature for 24 h and pretreating serum placed in autosampler for 36 h. The average bias was -4.09% between LC-MS/MS and the target value of EQA with a correlation formula of Y_LC-MS/MS=1.01X_{target value}-0.09, r=0.99. Electrochemiluminescence immunoassay results were 36.9% higher on average than those of LC-MS/MS. LC-MS/MS is simple and specific for the determination of serum digoxin. The results reported by LC-MS/MS are comparable with the national external quality assessment proficiency test. There is significant difference in digoxin results between LC-MS/MS and electrochemiluminescence immunoassay.

The detection and analysis of sperm motion parameters and DNA fragmentation index in asthenozoospermia patients

JIAO Ruibao, YAO Yuyou, TANG Jibin, FENG Hengxiao, FENG Junrong

2015, 30(5):  442-445.  DOI: 10.3969/j.issn.1673-8640.2015.05.008

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To study the changes of sperm motion parameters and DNA fragmentation index (DFI) in patients with asthenozoospermia. The routine analysis, semen analyzer activity detection and fluorescence microzooscopy of 41 cases of asthenozoospermia patients(asthenozoospermia group) and 48 cases of normal vitality patients(normal vitality group) were performed. Semen routine parameters(sperm concentration, total number of sperm and sperm activity rate), sperm motion parameters [progressive motility(PR), average path velocity(VAP), curvilinear velocity(VCL), straight line velocity(VSL), linearity(LIN), wobble(WOB), straightness(STR), amplitude of lateral head displacement(ALH), beat-cross frequency(BCF) and mean angular displacement(MAD)] and DFI in patients with asthenozoospermia and normal vitality group were compared. The sperm concentration, total number of sperm, sperm activity rate, PR, VAP, VCL, VSL, LIN, WOB, STR, ALH, BCF and MAD of asthenozoospermia group were (69.0±49.1)×10⁶/mL, (203±159)×10⁶, 60.8%±15.7%, 21.1%±8.0%,(10.7±2.8)μm/s, (20.6±4.9)μm/s, (6.3±2.2)μm/s, 29.9%±8.1%,51.4%±5.8%, 57.2%±10.4%, (0.53±0.12)μm, (3.20±0.85)Hz and (14.2±3.2)°, respectively. These parameters were significantly lower than those of normal vitality group[(98.8±38.2)×10⁶/mL(P<0.05),(281±171)×10⁶(P<0.05), 82.0%±7.9%(P<0.01), 45.1%±8.5%(P<0.01), (18.5±3.0)μm/s(P<0.01), (30.7±5.3)μm/s(P<0.01), (12.0±2.3)μm/s (P<0.01), 39.6%±7.0%(P<0.01), 60.5%±5.0%(P<0.01), 65.1%±7.2%(P<0.01), (0.72±0.13)μm (P<0.01), (5.07±0.81)Hz(P<0.01) and (17.8±2.5)°(P<0.01)]. The DFI in asthenozoospermia group(17.9%±12.5%) was significantly higher than that in normal vitality group(8.5±6.4%, P<0.01). Besides the performance of low sperm motility, asthenozoospermia is still accompanied by decreasing sperm concentration, total number of sperm and sperm motion parameters and increasing sperm DFI. The change of these parameters may be one of the mechanisms of male infertility caused by asthenozoospermia.

Cell morphology and immunophenotype in 12 patients of acute bilineage leukemia

FAN Liquan, CHEN Weiqin, WANG Jianbiao, SHENG Yan, WU Jing

2015, 30(5):  446-449.  DOI: 10.3969/j.issn.1673-8640.2015.05.009

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To investigate the relationship between cell morphology and immunophenotype of acute bilineage leukemia(aBLL). A total of 12 aBLL patients were enrolled, and the marrow cell morphology and histochemistry staining were performed. The immunophenotype was detected, including HLA-DR, CD34, MPO, CD13, CD33, CD11b, CD117, CD14, CD64, CD3, CD4, CD7, CD10, CD19, CD20, CD22 and CD79a. There were not only some differences but also complementaries between FAB and immunophenotype classification of aBLL. Of the 12 aBLL patients, 6 cases were diagnosed by cell morphology as acute myeloid leukemia (AML) [M2(4 cases) and M1(2 cases)], 2 cases as acute mixed lineage leukemia (AMLL) and 4 cases as ALL-L2, respectively. The 12 cases were diagnosed by immunophenotype classification as aBLL, while 10 cases were myeloid-B lymphocyte mix (My-B), and 2 cases were myeloid-T lymphocyte mix(My-T). aBLL is a rare type of leukemia. The complementarity between immunophenotype and cell morphology can effectively reduce the misdiagnosis and missed diagnosis of aBLL.

Analysis on the expressions of serum IgE, FcεRI and their antibodies in patients with benign thyroid diseases

WANG Yuping, CUI Zelin, LIN Lihui, WANG Juan, LI Jia, PENG Xia, XIE Guogang, LI Li

2015, 30(5):  450-453.  DOI: 10.3969/j.issn.1673-8640.2015.05.010

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To analyze the expressions of immunoglobulin E (IgE), FcεRIα and their antibodies in serum from patients with benign thyroid diseases and investigate their relationships with benign thyroid diseases. A total of 41 Graves' disease (GD) patients, 36 Hashimoto's thyroiditis (HT) patients, 36 thyroid nodule (TN) patients and 60 healthy subjects were enrolled. The expressions of FcεRIα, anti-IgE antibody and anti-FcεRI antibody were determined by competitive enzyme-linked immunosorbent assay (ELISA), and the level of IgE was determined by rate nephelometry. Their differences in 4 groups and their relationships with benign thyroid diseases were analyzed. The levels of IgE in GD, HT and TN groups had no statistical significance compared with that in healthy control group (P>0.05). The serum levels of FcεRIα and anti-IgE antibody in GD, HT and TN groups were higher than that in healthy control group (P<0.05). The level of anti-FcεRI antibody in GD group was higher than that in healthy control group. The relationships of the serum levels of IgE, FcεRIα, anti-IgE antibody and anti-FcεRI antibody with thyroid hormone autoantibody (TRAb) were not observed in HT or TN group, but there was a negative correlation of serum FcεRIα with TRAb (r_s=-0.350, P<0.05), and there was a positive positive correlation of anti-IgE and anti-FcεRI antibodies with TRAb in GD group (r_s=0.546 and 0.520, P<0.05). There are high expressions of serum IgE, FcεRIα and their antibodies in patients with benign thyroid diseases and a significant correlation of them with TRAb in GD group. The results suggest that these antibodies may participate in the occurrence and progress of benign thyroid diseases.

Expression of Fas in peritoneal fluid mononuclear cells among endometriosis patients and its significance

LIU Hongzheng, WANG Yuliang

2015, 30(5):  454-456.  DOI: 10.3969/j.issn.1673-8640.2015.05.011

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To investigate the relationship of Fas expression in peritoneal fluid mononuclear cells and endometriosis. A total of 55 patients with histopathologically diagnosed endometriosis after operation were enrolled as endometriosis group, and 52 patients without endometriosis were enrolled as control group. The peritoneal fluid mononuclear cells were isolated, the expression levels of Fas mRNA were determined by reverse transcriptase-polymerase chain reaction(RT-PCR), and the percentage of Fas expression cells were quantitatively detected by flow cytometry. Fas mRNA and the percentage of Fas expression cells were significantly higher in endometriosis group than those in control group(P<0.05). There was no statistical significance between stage Ⅰ-Ⅱand stage Ⅲ-Ⅳ(P>0.05). The abnormal Fas expression in peritoneal fluid mononuclear cells may play an important role in endometriosis.

The significance of allergen detection for eczema, allergic rhinitis and asthma patients

YAO Rongfeng, JIANG Peihong, XU Guoxiang, XU Long, LI Zhi, XUE Long, ZHAO Di

2015, 30(5):  457-460.  DOI: 10.3969/j.issn.1673-8640.2015.05.012

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To understand allergen IgE antibody distribution for different allergic diseases, in order to provide the reference for clinical prevention and treatment. A total of 236 patients with allergic rhinitis, 250 patients with asthma and 275 patients with eczema were enrolled. Western blot was performed to detect the serological specific allergen IgE antibody. The allergens for allergic rhinitis, asthma and eczema were analyzed comparatively. The inhaled allergen detection rate was 41.66% in the 761 patients, and the ingested allergen detection rate was 8.28%. Dust mite(43.10%) was the most common allergen, and dog hair(12.22%), milk(11.43%) and house dust(10.78%) were the other 3 common allergens. The prevalence rates of dust mite(62.35%), dog hair(21.11%), house dust(18.89%) and milk(26.47%) in 11-30 years old group were higher than those in the other age groups(P<0.05). The allergen detection rates of cockroach, milk and Penicillium notatum/Branch spore mould /Aspergillus fumigatus/Aspergillus niger/Streptomyces josamycetinus in eczema patients were obviously higher than those in allergic rhinitis and asthma patients(P<0.05). There was no statistical significance in allergen distribution of allergic rhinitis patients and asthma patients(P>0.05). Serum allergen detection might be conducive to identify the allergic status of patients, and provides a reliable reference to assist in the diagnosis of allergic diseases, prevent from environmental factors and make treatment plan.

Research on the diagnosis significance of human epididymis protein 4 for chronic kidney disease

WU Ping, YE Weimin, LI Chen, XIA Xiaohong, ZHANG Zhencheng, ZHAO Wenjing, CUI Yanfang, ZHANG Caiying, ZHONG Renqian, YANG Zaixing

2015, 30(5):  461-464.  DOI: 10.3969/j.issn.1673-8640.2015.05.013

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To determine the level of human epididymis protein 4(HE4), and to evaluate its diagnosis significance for chronic kidney disease(CKD). A total of 81 CKD patients and 73 healthy controls were enrolled. The determinations of serum HE4, carbohydrate antigen(CA)125, alpha fetoprotein(AFP), carcinoembryonic antigen(CEA), CA199, CA15-3 and so on were performed by electrochemiluminescence. The results were analyzed by receiver operating characteristic(ROC) curve for the diagnosis of CKD with different stages. The levels of serum HE4, CA125, CEA, CA15-3, urea, creatinine and uric acid were significantly higher in CKD patients than those in healthy controls(P<0.05), while there was no statistical significance for AFP and CA199 between the 2 groups (P>0.05). ROC curve analysis indicated that compared with other parameters, HE4 had a better diagnosis significance for CKD. The area under the curve of HE4 was larger than those of urea, creatinine and uric acid. Additionally, there were statistical significances for serum levels of HE4, CEA, urea, creatinine and uric acid among various stages of CKD(P<0.005). Moreover, the serum levels of these parameters increased with severity of CKD. ROC curve analysis showed that serum HE4, urea, creatinine and uric acid had significant diagnosis significance for staging CKD(CKD4-5), with the area under the curve>0.9. Serum HE4 shows an excellent diagnosis significance for CKD. Furthermore, it is closely associated with the severity of CKD. It is significant for judging the stages of CKD.

Clinical significance of the combined detection of GAD-Ab, IA2-Ab and ICA in patients with diabetes mellitus

ZHOU Houqing, WU Xinggui

2015, 30(5):  465-467.  DOI: 10.3969/j.issn.1673-8640.2015.05.014

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To investigate the clinical significance of the combined detection of anti-glutamic acid decarboxylase antibody(GAD-Ab), anti-protein tyrosine phosphatase antibody(IA2-Ab) and anti-islet cell antibody(ICA) in the diagnosis of diabetes mellitus. A total of 136 patients with diabetes mellitus were enrolled, including 54 cases of type 1 diabetes mellitus and 82 cases of type 2 diabetes mellitus, and 65 healthy controls were enrolled as healthy control group. GAD-Ab and IA2-Ab were determined by enzyme-linked immunosorbent assay (ELISA), and ICA was determined by indirect immunofluorescence assay. The positive rates of ICA, GAD-Ab and IA2-Ab in patients with type 1 diabetes mellitus(22.2%, 61.1% and 11.1%) were significantly higher than those in patients with type 2 diabetes mellitus (2.4%, 4.9% and 1.2%) and control group (0%, 0% and 0%, P<0.05). The positive rate of GAD-Ab in patients with type 1 diabetes mellitus was higher than those of ICA and IA2-Ab(P<0.001). The sensitivity and diagnostic accordance rate of GAD-Ab were higher than those of ICA and IA2-Ab(P<0.05). The sensitivities and diagnostic accordance rates of the combined detections of GAD-Ab+ICA, GAD-Ab+IA2-Ab and ICA+GAD-Ab+IA2-Ab were also higher than those of ICA and IA2-Ab(P<0.05). The combined detection of GAD-Ab, IA2-Ab and ICA has important significance in the diagnosis and classification of diabetes mellitus.

Study on the drug resistance and in vitro adhesion ability difference of Acinetobacter baumannii with different genotypes

TA La, WANG Junrui, CUI Jinghua, DU Xiaoli, YANG Limin, SUN Peng, WEI Changmei, ZHANG Junli

2015, 30(5):  468-473.  DOI: 10.3969/j.issn.1673-8640.2015.05.015

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To analyze the drug resistance characteristics and in vitro adhesion abilities of Acinetobacter baumannii with different genotypes, to provide the reference for further study on the relationship between the evolution of drug resistance of Acinetobacter baumannii and its adhesion ability change, and to guide infection prevention and control. Pulsed field gel electrophoresis(PFGE) was used to do molecular typing for 50 isolates of Acinetobacter baumannii from different inpatients in Inner Mongolia. In vitro adhesion abilities of Acinetobacter baumannii with different genotypes were detected by fibronectin adherence assay. The 50 isolates had 22 PFGE genotypes, among which A and E types were the advantageous types, and the rates were 22%(11/50)and 24%(12/50), while the isolates with L type were resistant to all tested antibiotics, and the drug resistance of the isolates belonging to the remaining genotypes were significantly different. The isolates of A type were mainly isolated from 2 regions, Xing'an Meng and Chifeng City. The isolates of E type were all isolated from Hohhot. Fibronectin adherence assay showed that the isolates with G type and J type showed the highest in vitro adhesion ability, while the isolates with J type were sensitive to all tested antibiotics, and the isolates with D type showed the lowest in vitro adhesion ability, only being resistant to carbapenem. The genotypes of Acinetobacter baumannii isolated from Inner Mongolia show diverse features, which seems to be specific with different districts. There is no significant correlation between the drug resistance acceleration of Acinetobacter baumannii and its in vitro adhesion abilities.

Analysis of drug resistance of multi-drug resistant and extensively-drug resistant Gram negative bacillus from the elder patients with nosocomial infection in Huadong Hospital

FANG Yi, ZHAO Fuju, ZHAO Hu

2015, 30(5):  474-477.  DOI: 10.3969/j.issn.1673-8640.2015.05.016

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To guide the reasonable antibiotic clinical use and provide the reference for effective prevention and nosocomial infection control by understanding the drug resistance and distribution in elder patients infected with multi-drug resistant and extensively-drug resistant Gram negative bacillus in Huadong Hospital. The data of 228 elder patients infected with multi-drug resistant bacillus and 42 patients infected with extensively-drug resistant bacillus were studied retrospectively(all the elder patients were over 60 years old). Bacterial culture, identification and drug susceptibility were performed for the specimens from patients(sputum, blood, urine, bile, wounds secretion and so on). The resistant genes of 8 extensively-drug resistant Acinetobacter baumannii were determined by polymerase chain reaction(PCR). The rank of the multi-drug resistant bacillus was Acinetobacter baumannii, Pseudomonas aeruginosa and Escherichia coli, as well as the rank of the extensively-drug resistant bacillus was Acinetobacter baumannii, Pseudomonas aeruginosa and Burkholderia cepacia. The TEM, SHV, PER, ant(2″)-Ⅰ, ant(3″)-Ⅰ, ant(6')-Ⅰb, OXA-23, gyrA and qacEΔ1 genes were detected in the extensively-drug resistant Acinetobacter baumannii. The results of IMP, VIM and OXA-24 genes were negative. Since the severe drug resistance of Acinetobacter baumannii and Pseudomonas aeruginosa from elder patients in Huadong Hospital, especially in the department of respiration and intensive care unit, we should effectively prevent, delay and control the generation and spreading of resistant isolates by strengthening the reasonable antibiotic use as well as monitoring the resistant isolates.

Comparison analysis of microbiological monitoring between hemodialysate and reverse osmosis water under different media

ZHOU Yuechang, REN Lina, WANG Xuhui, CHEN Jianwen, CHEN Miaopei, MAO Hongzhong, HAN Lijie

2015, 30(5):  478-483.  DOI: 10.3969/j.issn.1673-8640.2015.05.017

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To compare the difference between media, incubation temperatures and culture times on the choice of hemodialysate and reverse osmosis water for microbiological monitoring of heterotrophic plate count, and further to confirm the best culture method of microbiological monitoring for hemodialysate and reverse osmosis. The polluted samples were simulated by using sterile dialysate and sterile reverse osmosis water, and then were incubated in 25℃ and 35℃ with eosin methylene blue agar(EMB), tryptone soy agar(TSA) and tryptone glucose extract agar(TGEA)for 48 h, 72 h and 7 d, respectively. Either on 25℃ or 35℃, the bacteria's growth condition in TGEA was better than those under other conditions(P<0.05). The colony numbers of 72 h incubation were more than those of 48 h incubation in all media(P<0.05). However, there was no statistical significance of colony numbers between 7 d incubation and 72 h incubation(P > 0.05). The microbiological monitoring for hemodialysate and reverse osmosis water needs to use pour plate method with TGEA, incubating in 25℃ and 35℃ for 72 h, which may provide the effective reports for ensuring the safety of hemodialysis patients.

Relationships of oxidized low-density lipoproteins and C reactive protein with in-stent restenosis after PCI

HE Wenjun, HUANG Jiaping, LIANG Weidong, MO Xiang, LIANG Shaohua

2015, 30(5):  484-488.  DOI: 10.3969/j.issn.1673-8640.2015.05.018

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To investigate the predictive significance of oxidized low-density lipoproteins(ox-LDL) and C reactive protein(CRP)for in-stent restenosis(ISR)after percutaneous coronary intervention(PCI). A total of 186 patients who were given PCI and followed up for 6 months were enrolled. Of them, 22 patients had ISR(ISR group), while the other 164 patients had not ISR(no-ISR group). The pre- and post-PCI serum ox-LDL and CRP were detected. There was no statistical significance between the 2 groups in terms of age, sex, morbidity, serum lipid and vessel lesion(P>0.05). Serum ox-LDL and CRP increased significantly in ISR group at post-PCI compared with pre-PCI (P<0.05), while there was no significant change in no-ISR group (P>0.05). Serum ox-LDL were also higher in ISR group compared with no-ISR group at pre- and post-PCI(P<0.05). There was no statistical significance for CRP level between the 2 groups at pre-PCI (P>0.05). However, the significant increase of CRP level in ISR group compared with no-ISR group was present at post-PCI(P<0.05). Correlation analysis revealed that there was positive correlation between pre- and post-PCI for serum ox-LDL and CRP in both ISR and no-ISR groups [ISR group: r(P) values of pre- and post-PCI were 0.392(0.020) and 0.431(0.010); no-ISR group: r(P) values of pre- and post-PCI were 0.382(0.024) and 0.526(0.001)]. The Logistic regression analysis showed that post-PCI ox-LDL[odds ratio (OR)=1.27, 95% confidence interval(CI): 1.05-1.79]and pre-PCI CRP(OR=1.82,95%CI:1.68-2.30)were independent risk factors of ISR. Post-PCI ox-LDL and pre-PCI CRP have certain significance in the prediction of ISR.

Correlation between serum TSH concentrations and cardiovascular severity in patients undergoing PCI

WU Weiyun, WU Shengchao, YAN Hongmei, WU Jiong, GUO Wei, ZHANG Chunyan, SONG Binbin, PAN Baishen

2015, 30(5):  489-494.  DOI: 10.3969/j.issn.1673-8640.2015.05.019

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To investigate the correlation between serum thyroid-stimulating hormone(TSH)concentrations and cardiovascular severity in patients undergoing percutaneous coronary intervention(PCI). Of 1 122 patients undergoing PCI, 1 000 who met inclusion criteria were classified into 3 groups according to serum TSH concentrations: normal,subclinical hypothyroidism and subclinical hyperthyroidism groups. The main outcomes were Gensini score with Gensini scores >50 defined as high cardiac risk and the percentage undergoing stent implantation. The percentages of patients with Gensini scores >50 differed significantly in normal and subclinical hypothyroidism groups(P=0.003). Binary Logistic regression analysis showed that subclinical hypothyroidism was an independent risk factor for cardiovascular disease[odds ratio (OR)=1.855, 95% confidence interval (CI): 1.244-2.766, P=0.002]. In contrast,Gensini score >50 and stent implantation did not correlate with TSH concentration in normal group. Subclinical hypothyroidism is a risk factor for cardiovascular disease in patients undergoing PCI. The correlation between normal TSH concentrations and cardiovascular disease requires further investigation.

Evaluation of BNP quantification by single-epitope sandwich combined with cyclic enhanced immunofluorescent assay

XIAO Chunhai, LIANG Shuang, LIU Jingfan, LI Feifei, ZHU Qinghua, QU Peipei, DONG Zhiwu

2015, 30(5):  495-499.  DOI: 10.3969/j.issn.1673-8640.2015.05.020

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To evaluate B-type natriuretic peptide quantification by single-epitope sandwich(SES)combined with cyclic enhanced immunofluorescent assay(CEIFA). By SES combined with CEIFA, BNP was determined. The precision, linear range, function sensitivity, recovery rate and sample stability were evaluated, and 45 samples were determined by the method, which the results were evaluated with those of SIEMENS ADVIA Centaur CP chemiluminescence analyzer (Centaur CP). The within-run and between-run coefficients of variation(CV) were 4.38%-7.84% and 10.47%-11.21%, respectively. The linear range was 25-5 000 pg/mL. The function sensitivity was 12.5 pg/mL. The recovery rate was 95.04%-109.2%. The correlation formula between the novel method and Centaur CP was Y=0.998 3X-22.38 (r=0.969, P<0.01). The request of sample stability of SES combined with CEIFA was lower than that of Centaur CP. SES combined with CEIFA for BNP quantification has a good performance, and there are some advantages compared with traditional double-antibody sandwich chemiluminescence assay.

Research on a quantitative method to detect viable Salmonella by PMA-qPCR in livestock and poultry meat

YU Ying, WANG Wenjing, LU Ye

2015, 30(5):  500-506.  DOI: 10.3969/j.issn.1673-8640.2015.05.021

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To enumerate Salmonella in meat of livestock and poultry rapidly and accurately by using propidium monoazide(PMA) combined with real-time fluorescence quantitation polymerase chain reaction(qPCR). The light exposure time and the concentration of PMA were optimized to establish PMA-qPCR. The standard curve was established by standard plasmid. The sensitivity and specificity were investigated. This method was used for the quantitation determination of Salmonella in livestock and poultry meat. The amplification of DNA derived from Salmonella dead cells could be inhibited without affecting the viable cells when PMA was at a dose of 15 μg/mL and exposed for 5 min. The cycle threshold values(Ct) and standard plasmid model cell copy number presented the satisfactory linear, and the correlation coefficient r² approached 0.997 9. This method could detect as low as 10 copies/reaction. The minimum detection level was 21 copies/μL by PMA-qPCR. In artificial chicken samples, PMA-qPCR could detect as low as 10³ CFU/mL. It was possible to quantify viable Salmonella in meat of livestock and poultry by PMA-qPCR.

Influence of 5-Aza-CdR on the apoptosis of lung cancer A549 cell and the expression of FANCF gene

YU Zongtao, ZHANG Jicai, GAO Qiong, GAO Bo, HU Chunhui

2015, 30(5):  507-511.  DOI: 10.3969/j.issn.1673-8640.2015.05.022

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To investigate the influence of 5-Aza-2'-deoxycytidine(5-Aza-CdR) on the apoptosis of lung cancer A549 cell and the expression of Fanconi anemia complementation group F(FANCF) gene. A549 cells were treated with 5-Aza-CdR(0.5, 5 and 50 μmol/L, respectively). The growth of A549 cells was observed by 3-(4,5-dimethylthiazol)-3,5-diphenyltetrazolium bromide(MTT) assay. The methylation status of FANCF gene was observed by methylation specific polymerase chain reaction(PCR). The expression of FANCF mRNA was observed by fluorescence quantitation PCR. The apoptosis rate of A549 cells was analyzed by flow cytometry. A549 cells treated with 5-Aza-CdR displayed a slow growth. The rate of cell proliferation inhibiting (CPIR) for A549 cells changed with the concentration and treatment time of 5-Aza-CdR (P<0.005, P<0.001). FANCF mRNA expression increased after treatmert. The apoptosis rates after treatment had a positive correlation with 5-Aza-CdR dose (r=0.998, P<0.05). 5-Aza-CdR can induce the apoptosis of A549 cells by inducing demethylation and thereby enchancing FANCF gene, enhancing tumor suppressor function, but it can increase the risk of resistance to cisplatin.

Uncertainty evaluation on the reference method of leukocyte counts for fresh blood

GUO Xiaojun, WANG Qing, SONG Ying, MAIO Yingbo, XU Lei

2015, 30(5):  512-516.  DOI: 10.3969/j.issn.1673-8640.2015.05.023

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To study the evaluation method and procedure on the uncertainty of leukocyte counts by reference method for fresh blood. The reference method recommended by the International Committee for Standardization in Hematology(ICSH) was used for leukocyte counts. The components of uncertainty were validated. The International Organization for Standardization(ISO) Guide to the Expression of Uncertainty of Measurement (GUM), type A and type B methods were used to evaluate each component. Combined standard uncertainty and expanded uncertainty were calculated according to 95% confidence interval. The leukocyte counts from fresh blood had the average value of 6.71×10⁹/L. The combined standard uncertainty was 0.097×10⁹/L(95% confidence interval, coverage factor k=2), and the expanded uncertainty was 0.19×10⁹/L. The main sources of uncertainty are from the overlapped calculation correction and pipetting volume. Therefore, there should be strictly control on pipetting volume in order to make lower uncertainty. The evaluation model of uncertainty is established, which can be applied to the leukocyte counts and standardization, providing effective methods for traceability.

Analysis on out-of-control items during the internal quality control on clinical biochemical determination in Department of Clinical Laboratory, Inner Mongolia Forestry General Hospital

WANG Xiaoyan, SUN Hui, SUN Gang, CAI Shumin

2015, 30(5):  517-519.  DOI: 10.3969/j.issn.1673-8640.2015.05.024

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To analyze out-of-control reasons of clinical biochemical internal quality control from January 2009 to December 2013 in Department of Clinical Laboratory, Inner Mongolia Forestry General Hospital, to summarize the corrective and preventive measures, and to reduce out-of-control rate gradually. According to the reference of Westgard quality control rules, the out-of-control results of daily internal quality control by OLYMPUS AU5421/AU640 automatic biochemical analyzer and original reagents, control materials and calibrators were analyzed retrospectively for 5 years. In terms of calibrators, reagents, human factors, control materials, instruments and other factors, the statistical analysis about out-of-control reasons was performed. For 5 years, there were 242 out-of-control cases, including 69 calibration problems, 54 reagent problems, 50 control material problems, 31 human factor problems, 23 instrument problem and 15 other problems. Calibration, reagent, control material reasons accounted for more than 70% of all reasons, and compared with human factor, instrument and other reasons, there was statistical significance(P<0.05). Calibrators, reagents, human factors, control materials and instruments are the main reasons causing out-of-control situations. Clinical laboratories should take effective measures, regulate the standard operating procedure, strictly control the quality of biochemical determination and persist in improving the quality of clinical inspection.

Investigation on the establishment of reference change values for test results

CHEN Hongyan, QIU Haiwen

2015, 30(5):  529-532.  DOI: 10.3969/j.issn.1673-8640.2015.05.028

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Reference change values (RCV) are the random variations of 2 test results of the total. Through the evaluation on the statistical significance of individual difference of continuous test results, the clinical monitoring of patients' test results can be performed. Medical laboratory technicians should establish reference change values correctly on the basis of full understanding of theory and guide the clinical application.

The research progress of early warning biomarker for sepsis

XU Cheng, XU Yuanhong

2015, 30(5):  533-536.  DOI: 10.3969/j.issn.1673-8640.2015.05.029

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Sepsis is a kind of systemic inflammatory response syndromes(SIRS) induced by infection, which has been clinically confirmed the presence of bacteria or highly suspicious infections. More and more researchers have paid attention to the early diagnosis of sepsis recently. This review has focused on the capabilities of cytokine, procalcitonin(PCT), endotoxin, high-sensitivity C reactive protein(hs-CRP), serum amyloid protein A(SAA), heparin-binding protein(HBP), (1,3)-beta-D-glucan(BG) and galactomannan(GM) to diagnose sepsis early and assess the severity and prognosis, in order to provide new thoughts for the early diagnosis and treatment of sepsis.

Research progress of apolipoprotein E receptor 2

LU Donghe, FU Binli, GAO Fei, ZHANG Wenjing, LIU Yanhong

2015, 30(5):  537-540.  DOI: 10.3969/j.issn.1673-8640.2015.05.030

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Apolipoprotein E receptor 2(ApoER2),a kind of cell surface glycoprotein, widely existing in many tissues of body cells, can interact with a variety of different structures and functions of ligand, and plays an important role in signal transduction. This paper mainly reviews the research progress about the structure of ApoER2 and its biological function, ApoER2 expression and relative diseases.

The clinical application significance of serum total bile acid detection in intrahepatic cholestasis of pregnancy

LIU Jintao

2015, 30(5):  541-544.  DOI: 10.3969/j.issn.1673-8640.2015.05.031

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Intrahepatic cholestasis of pregnancy(ICP) can increase the risk of adverse pregnancy outcome, and can seriously affect the prognosis of perinatal infants, and it is one of the main perinatal mortality factors. ICP should be early diagnosed and aggressively treated to reduce perinatal morbidity and mortality. Serum total bile acid(sTBA) is a sensitive index for the early diagnosis and treatment of ICP. This paper reviews the relationship of sTBA with ICP and sTBA with liver function indices, the application significance for ICP, the influence of sTBA on fetus and the pathogenesis of ICP, and reviews the role of sTBA in ICP and its clinical application significance.