Laboratory Medicine

Clinical mass spectrometry：principles of mass spectrometry for clinical laboratories

ZHANG Yan, Thomas Jackson

2018, 33(1): 1-9. DOI: 10.3969/j.issn.1673-8640.2018.01.0001

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Mass spectrometry （MS） is a fast growing area in clinical laboratories due to its exceptional specificity,high sensitivity and ability to detect multiple compounds simultaneously. Several types of mass spectrometers are being used in clinical laboratories globally. As a result,tremendous improvements in assay performance are in process. This review is intended as a basic introduction to MS and describes the history of MS in clinical laboratories,instrumentation,ionization techniques and sample preparation. The review also discusses challenges to implementation in clinical laboratories and discussions on future instrumentation and future uses of MS.

Establishment and verification for the reference intervals of sperm concentration and sperm total motility by different determination systems in Guangzhou

SHI Hanzhen, CHEN Xi, ZENG Dongyu

2018, 33(1): 10-14. DOI: 10.3969/j.issn.1673-8640.2018.01.002

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Objective To establish the reference intervals of semen routine analysis's main indicators （sperm concentration and sperm total motility）according to regional characteristics and different determination systems,and to verify whether they are suitable for Guangzhou local population. Methods According to the Clinical and Laboratory Standards Institute （CLSI） C28-A3 document,145 healthy subjects were enrolled. Computer assisted sperm analysis（CASA）, Marker plate and improved NiuBao plate were used. The reference intervals of 3 different determination systems were established. Another 20 cases of healthy subjects were enrolled to verify the applicability of these reference intervals. Results The reference intervals for sperm concentration were ≥27.6×10⁶/mL for CASA,≥35.4×10⁶/mL for Marker plate and ≥31.0×10⁶/mL for improved NiuBao plate. The reference intervals for sperm total motility were ≥40.9% for CASA and ≥49.4% for Marker plate. The reference intervals for sperm forward motility were ≥32.2% for CASA and ≥35.0% for Marker plate.Conclusions The established reference intervals are applicable for our clinical laboratory and local population,even they can be used for other clinical laboratories in local area.

NT-proBNP,cTnI,Hcy,DD and hs-CRP for the diagnosis of coronary heart disease by ROC curves

YANG Yan, ZHANG Fujun, SHEN Songkun, WANG Houzhao

2018, 33(1): 15-19. DOI: 10.3969/j.issn.1673-8640.2018.01.003

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Objective To evaluate the roles of N-terminal pro-B-type natriuretic peptide（NT-proBNP）,cardiac troponin I（cTnI）,homocysteine（Hcy）,D-dimer（DD） and high-sensitivity C-reactive protein （hs-CRP） for the diagnosis of coronary heart disease by receiver operating characteristic （ROC） curves. Methods A total of 87 patients with coronary heart disease were enrolled. There were 31 cases with acute myocardial infarction （AMI）,26 cases with unstable angina pectoris （UAP） and 30 cases with stable angina pectoris （SAP）. Totally,40 healthy subjects were enrolled as healthy control group. The levels of NT-proBNP and cTnI were determined by time-resolved immunofluorescence,DD and hs-CRP were determined by immunoturbidimetric assay,and Hcy was determined by enzymatic cycling assay. ROC curves were used to analyze optimal cut-off values,areas under curves（AUC）,sensitivities and specificities of NT-proBNP,cTnI,Hcy,DD and hs-CRP. Results The levels of NT- proBNP,cTnI,Hcy,DD and hs-CRP in coronary heart disease group were higher than those in healthy control group（P<0.05）. The increases of NT-proBNP,cTnI,Hcy,DD and hs-CRP from low to high in turn were SAP group,UAP group and AMI group（P<0.05）. The level of hs-CRP in SAP group was higher than that in healthy control group （P<0.05）,and there was no statistical significance for the other 4 indicators between the 2 groups（P>0.05）. The AUC of NT-proBNP,cTnI,Hcy,DD and hs-CRP in diagnosing coronary heart disease were 0.961,0.855,0.800,0.842 and 0.878,respectively. The optimal cut-off value of NT-proBNP was 321.00 ng/L,the sensitivity was 85.1%,and the specificity was 100.0%.Conclusions NT-proBNP,cTnI,Hcy,DD and hs-CRP can be used as valuable indicators in the diagnosis of coronary heart disease,and NT-proBNP is the most valuable.

Relations of serum alanine aminotransferase and gamma-glutamyltransferase with type 2 diabetes mellitus

LIU Wenhui, BAI Jin'e, ZHANG Hui

2018, 33(1): 20-25. DOI: 10.3969/j.issn.1673-8640.2018.01.004

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Objective To investigate the relations of serum alanine aminotransferase（ALT）and gamma-glutamyltransferase（GGT） with type 2 diabetes mellitus（T2DM）,and to provide a reference for the early prevention of T2DM. Methods A total of 2 597 healthy subjects, who finished questionnaires,were enrolled. They were classified into 4 groups according to the quartiles of serum ALT and GGT levels（Q1,Q2,Q3 and Q4 groups from low levels to high levels）. Logistic regression analysis was used to evaluate the relations of serum ALT and GGT with T2DM. Results Body mass index（BMI）,systolic blood pressure（SBP）, diastolic blood pressure（DBP）, glucose（Glu）,total cholesterol（TC）, triglyceride（TG）,low-density lipoprotein cholesterol（LDL-C）and the prevalence rate of hypertension increased with serum ALT or GGT（P<0.05）, while high-density lipoprotein cholesterol（HDL-C）decreased with serum ALT or GGT（P<0.001）. For serum ALT and GGT, the risks of T2DM in Q4 group had 1.70 times [odds ratio（OR）=1.70,95% confidence interval（CI）1.05-2.73] and 2.38 times（OR=2.38,95%CI 1.77-3.17）higher than those in Q1 group, respectively, after the adjustment of age,sex,smoking,drinking,physical activity,family history of diabetes mellitus,BMI,TG,TC,HDL-C,LDL-C and hypertension.Conclusions Serum ALT and GGT are related with the risk of T2DM.

Relationship between anti-carbamylated protein antibody and rheumatoid arthritis

CHEN Yiqiong, ZHAO Di, REN Fang, DENG Xiaogang, XU Long, LI Zhi

2018, 33(1): 26-30. DOI: 10.3969/j.issn.1673-8640.2018.01.005

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Objective To analyze the level of anti-carbamylated protein （Carp） antibody in patients with rheumatoid arthritis （RA）,and to investigate the role of anti-Carp antibody in the diagnosis and evaluation of RA. Methods The levels of serum anti-Carp IgG and anti-Carp IgA antibodies in RA group （102 cases）,non-RA group （55 cases） and healthy control group （83 cases） were determined by enzyme-linked immunosorbent assay（ELISA）. The difference of the 2 indices among the 3 groups and the relationship between anti-Carp antibody and RA were evaluated. Results The level of anti-Carp IgG antibody in serum of RA group was （8.60±2.64）U/L,and the positive rate was 42.16%. The level of anti-Carp IgA antibody in serum of RA group was （6.65±3.09）U/L,and the positive rate was 46.08%,which were higher than those in non-RA group and healthy control group （P<0.05）. The positive rate of anti-Carp IgG antibody was 46.67%,and the positive rate of anti-Carp IgA antibody was 33.33% in anti-cyclic citrullinated peptide （CCP） antibody negative RA patients. The levels of IgG,IgA and IgM in anti-Carp IgG antibody positive group had statistical significance compared with those in anti-Carp IgG antibody negative group （P<0.05）,and the levels of IgG,IgA,IgM and complement （C）4 in anti-Carp IgA antibody positive group were higher than those in anti-Carp IgA antibody negative group （P<0.05）. There was no correlation of anti-Carp IgG antibody with anti-CCP antibody and rheumatoid factor（RF）（P>0.05）,and there was no correlation of anti-Carp IgA antibody with anti-CCP antibody and RF （P>0.05）.Conclusions Anti-Carp antibody is a new serological marker for the diagnosis of RA,and it may be associated with the severity of the disease,which may contribute to the diagnosis of anti-CCP antibody negative RA.

Autoantibody-related parameters in patients with autoimmune diseases and healthy subjects

CHEN Shuimian, YU Chongzhao, LI Enling, ZHU Haoming, LU Jingbo, ZHENG Bing

2018, 33(1): 31-36. DOI: 10.3969/j.issn.1673-8640.2018.01.006

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Objective To investigate the roles of anti-nuclear antibody （ANA） and other related parameters for the diagnosis of different autoimmune diseases （AID）. Methods A total of 1 905 AID patients were enrolled and studied retrospectively. ANA and anti-extractable nuclear antigen （ENA）,anti-double-stranded DNA （ds-DNA） and anti-nucleosome （Nuc） antibodies were determined. Totally,4 433 healthy subjects were enrolled for ANA karyotypes and titer distribution. Th. Results were collected and analyzed by t test and non-parameter test,Spearman correlation analysis was used,and receiver operating characteristic （ROC） curve was used for evaluating diagnostic efficiency. Results The major patterns in primary biliary cirrhosis （PBC） and autoimmune hepatitis （AIH） were mixed type and cytoplasmic granules for ANA karyotypes,but there were homogenous, speckled and mixed patterns in other AID as well. Patients with ankylosing spondylitis （AS）,rheumatoid arthritis （RA） and inflammatory myositis （IM） had titer （median）≤1∶160,and other AID patients had high titer（≥1∶320）. For systemic lupus erythematosus （SLE） patients,the titers of ANA were positively correlated with anti-ds-DNA antibody （r=0.394,P<0.01）. In adult SLE group （898 cases）,the proportion of females,ANA and anti-ds-DNA antibody,anti-Nuc antibody and anti-uracil-1 low molecular weight ribonucleoprotein（U1RNP） antibody positive rates were higher than those in pediatric SLE group （103 cases,P<0.01）. When the titer was 1∶80,the sensitivity was 97.1%,and the specificity was 38.7%. When the titer was 1∶160,the sensitivity was 80.1%,and the specificity was 80.8%.Conclusions The positive rates and titers of ANA have difference among patients with different AID. Some special karyotype analysis can provide a reference for the diagnosis of AID. ANA titer is positively correlated with anti-ds-DNA antibody in patients with SLE. The titers of ANA （1∶80 and 1∶160）could be used for health examination screening and AID diagnosis.

Molecular epidemiological analysis of carbapenem-resistant Klebsiella pneumoniae

SHI Weiyang, LIU Yang, HAN Yichao, ZHENG Dandan, DAI Erkuan, ZHENG Bing, LI Min

2018, 33(1): 37-43. DOI: 10.3969/j.issn.1673-8640.2018.01.007

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： Objective To investigate the drug resistance and molecular epidemiology of carbapenem-resistant Klebsiella pneumoniae （CRKP） isolated from patients in Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine,and to provide a reference for clinical treatment and nosocomial infection control. Methods A total of 107 isolates of CRKP were collected from January 2013 to August 2015 in Renji Hospital. The susceptibilities of 107 CRKP isolates to 18 antimicrobial agents were determined by Kirby-Bauer disk diffusion method. The minimum inhibitory concentrations （MIC） of meropenem and tigecycline against 107 CRKP isolates were determined by microbroth dilution method. Carbapenem-resistant genes were determined by polymerase chain reaction （PCR）. The genotypes were identified by pulsed-field gel electrophoresis （PFGE）. Th. Results were analyzed by UPGMA clustering using NTSYS（2.1） software. Results The 107 CRKP isolates were susceptible to sulfamethoxazole （drug resistance rate 67.3%）,fosfomycin （drug resistance rate 24.0%） and tigecycline （drug resistance rate 100.0%） and were not susceptible to other 17 antimicrobial agents （drug resistance rate >95.0%）. KPC-2 gene was determined in all the 107 CRKP isolates. A total of 24 genotypes （A type-X type） were identified,and the 3 major genotypes were A type [19.6%（21/107）],B type [13.1%（14/107）] and C type [11.2%（12/107）]. There were 15 CRKP isolates of A type （15/21） collected within 1 month from departments of neurosurgery （6 isolates）,outpatient（3 isolates）,hematology （2 isolates）,geriatrics （2 isolates）,surgery intensive care unit （ICU）（1 isolate） and urology （1 isolate）. There were 4 CRKP isolates of F type （4/6） collected within 2 weeks from departments of general surgery （2 isolates） and geriatrics （2 isolates）. Other 22 genotypes were collected sporadically in different periods and different wards.Conclusions The major carbapenem-resistant gene of CRKP isolated from Renji Hospital is KPC-2. Outbreaks of A type and F type CRKP appear in Renji Hospital. CRKP isolates are susceptible to sulfamethoxazole,fosfomycin and tigecycline,and sulfamethoxazole is suggested as the first choice for treatment. Infection monitoring should be enhanced to reduce the spread of CRKP.

Roles of GM test and G test for the diagnosis of invasive pulmonary fungal infection and its clinical treatment efficiency

GAO Dongtian, SHEN Aihua

2018, 33(1): 44-49. DOI: 10.3969/j.issn.1673-8640.2018.01.008

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Objective To investigate the roles of galactomannan （GM） test and（1,3）-beta-D-glucan （G）test for patients with invasive pulmonary fungal infection （IPFI）. Methods The antigen levels of GM and G were determined by GM test and G test,respectively. The sensitivities,specificities,positive predictive values,negative predictive values,accuracies and missed diagnosis rates of GM test and G test were calculated. The information of IPFI patients,including age,department,concurrent disease,fungus species,correlation infection index and drug treatment efficiency,were analyzed retrospectively. Results There were 435 positive cases,and the proportions of males and females accounted for 63.0% and 37.0%,respectively. The age was （55.3±26.6）years old,and the patients over 50 years old accounted for 64.1%. The departments of respiratory,pediatrics and intensive care unit （ICU） accounted for 30.6%,17.5% and 14.2%,respectively. The proportions of severe pneumonia and chronic obstructive pulmonary disease （COPD） patients accounted for 31.5%. There were 88 isolates of Aspergillus fumigatus,36 isolates of Calbicans,12 isolates of Candida tropicalis and 6 isolates of Candida glabrata. The increase rates of C-reactive protein （CRP）,procalcitonin （PCT） and D-dimer （DD） were 77.0%,91.6% and 82.9%,respectively. The sensitivity,specificity,positive predictive value,negative predictive value,accuracy and missed diagnosis rate of GM test were 86.3%,93.0%,91.4%,88.7%,89.9% and 13.7%,and those of G test were 78.3%,77.2%,80.4%,74.9%,77.8% and 21.7%,respectively. The positive rates of GM test and G test were 34.7% and 42.3%（P>0.05）. The effective rates of voriconazole and caspofungin were 81.4% and 58.8%.Conclusions GM test and G test play roles in the clinical diagnosis of IPFI. The risk factors of IPFI include children,elderly male patients over 50 years old,severe pneumonia,COPD,blood diseases, rheumatic autoimmune disease,cerebral hemorrhage,cerebral infarction,uremia,tumor,Aspergillus fumigatus infection and Calbicans infection. Voriconazole and caspofungin are good treatment options.

Establishment of quantitative MAIPA assay for HPA-1a,HPA-3a and HPA-5b antibodies

LI Ruishu, LING Bing, LU Ping

2018, 33(1): 50-54. DOI: 10.3969/j.issn.1673-8640.2018.01.009

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Objective To establish quantitative monoclonal antibody-specific immobilization of platelet（PLT） antigen （MAIPA） assay for human PLT antigen（HPA）-1a,HPA-3a and HPA-5b antibodies.Methods MAIPA assay was applied on serial dilutions of standard or reference reagents containing antibodies against HPA-1a,HPA-3a and HPA-5b. Optimal experimental parameters were determined,and standard curves were constructed with linear regression. The sensitivity,specificity and reproducibility of the assay were evaluated. Results The optimal reagents and conditions for MAIPA assay were decided. The determination ranges were 0-4 IU,0-20 AU and 0-20 AU,and accuracies（recovery rates）were 96.20%-103.10%,97.90%-103.10% and 100.20%-106.10%. The within-run precisions [coefficients of variation （CV）] for HPA-1a,HPA-3a and HPA-5b antibodies were 1.3%-3.6%,2.7%-5.2% and 4.7%-5.3%. The between-run precisions （CV） were 4.0%-5.6%,4.4%-5.9% and 3.2%-6.3%. There was no positivity for anti-A,anti-B,anti-I and anti-P humanized antibodies with good specificities.Conclusions The established quantitative MAIPA assay for HPA-1a,HPA-3a and HPA-5b antibodies has good sensitivity,specificity,accuracy and precision. It plays a role in the clinical diagnosis of alloimmunization disease caused by HPA antibodies and safe transfusion of PLT.

Performance of double-antibody sandwich ELISA for chromogranin A determination

HU Yuyi, WU Hui, HAN Xu, ZHANG Chunyan, GUO Wei, LOU Wenhui, PAN Baishen

2018, 33(1): 55-59. DOI: 10.3969/j.issn.1673-8640.2018.01.010

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Objective To evaluate the performance of double-antibody sandwich enzyme-linked immunosorbent assay （ELISA）for chromogranin A （CgA）determination. Methods The precision,linearity and limit of determination of CgA determination by double-antibody sandwich ELISA were verified according to the Clinical and Laboratory Standards Institute （CLSI） EP15-A,EP6-A and EP17-A. The accuracy of CgA determination was assessed by manufacturer's quality control materials. A total of 66 neuroendocrine neoplasm patients,41 healthy subjects and 65 disease control patients were enrolled. The CgA levels in the 3 groups were determined,and receiver operating characteristic （ROC） curve was used to compare the efficiency of CgA determination in diagnosing neuroendocrine neoplasm. Results The within-run and between-run coefficients of variation （CV） of CgA determination by double-antibody sandwich ELISA were <14.00% and <8.00%,respectively. For accuracy evaluation,the relative bias was 4.51%,when the mean o. Results was within the indicated range. There was a good linear correlation（Y=0.793X-3.791,r²=0.988） in the range of 8-740 ng/mL. The limit of determination was 7.93 ng/mL. The difference of CgA levels in neuroendocrine neoplasm,healthy control and disease control groups had statistical significance （P<0.000 1）. The area under curve（AUC）of CgA determination in distinguishing healthy subjects and neuroendocrine neoplasm patients was 0.741,when the cut-off value was 47.18 ng/mL. The sensitivity was 78.8%,and the specificity was 68.3%.Conclusions CgA determination by double-antibody sandwich ELISA has good performance in precision,accuracy,linearity and limit of determination,which meets clinical needs. Serum CgA levels increase in patients with neuroendocrine neoplasm. CgA could be a marker for distinguishing healthy subjects and neuroendocrine neoplasm patients.

Consistency rate of UF-1000i automatic urine analyzer bacterial channel and mid-stream urine culturing

FENG Yuqing, HUANG Susu, LUO Jing, GU Guolong

2018, 33(1): 60-62. DOI: 10.3969/j.issn.1673-8640.2018.01.011

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Objective To study the consistency rate of UF-1000i automatic urine analyzer bacterial channel and mid-stream urine culturing,and to provide a reference for the early empirical use of antibiotics for urinary tract infection（UTI）. Methods Urine specimens were determined for bacterial culturing and urinary sedimentation analysis. Bacterial culturing was performed by Vitek 2 Compact. Urinary sedimentation analysis was performed by UF-1000i becterial channel. Results Th. Results of mid-stream urine culturing were as gold standard. For UF-1000i bacterial channel,the consistency rate,sensitivity and specificity of Gram-positive coccus were 90.7%,60.8% and 93.5%,and those of Gram-negative bacillus were 93.3%,76.7% and 96.8% （P<0.05）.Conclusions UF-1000i bacterial channel plays a role in the early clinical selection of antibiotics,especially in UTI caused by Gram-negative bacillus.

Influence of Del-1 on ICAM-1 expression of eosinophils in atopic allergy children

YAN Shu, ZHANG Hong

2018, 33(1): 67-71. DOI: 10.3969/j.issn.1673-8640.2018.01.013

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Objective To evaluate the influence of developmental endothelial locus-1 （Del-1） on intercellular cell adhesion molecule-1 （ICAM-1）expression of eosinophils in atopic allergy children.Methods Peripheral blood samples were collected from atopic allergy children （35 cases）,and eosinophils were isolated by magnetic-activated cell sorting （MACS）. ICAM-1 expression was determined by reverse transcription polymerase chain reaction （PCR） after administrating Del-1,anti-Del-1 antibody or interleukin 17 （IL-17）. Results Compared with control group treated with phosphate-buffered saline （PBS）,ICAM-1 expression was down-regulated after administrating Del-1（P<0.05）,and it was up-regulated after administrating anti-Del-1 antibody （P<0.05）. ICAM-1 expression was down-regulated after administrating Del-1 and IL-17 compared with that after administrating IL-17 only （P<0.05）. There was no statistical significance in non-eosinophils after administrating Del-1 or anti-Del-1 antibody （P>0.05）.Conclusions Del-1 can regulate the expression of ICAM-1,indicating that Del-1 can negatively regulate the intercellular adhesion of eosinophils,thereby limiting atopic allergy of children.

Six sigma quality management for choosing appropriate quality control rules in automatic hematological analyzer

MIAO Yingbo, SONG Ying, XU Lei, ZHU Peichao, WANG Qing

2018, 33(1): 72-75. DOI: 10.3969/j.issn.1673-8640.2018.01.014

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Objective To evaluate the performance of automatic hematological analyzer by six sigma（6σ） quality management,and to choose appropriate quality control rules with OPSpecs charts,monitor analyzer's performance and ensure determination quality. Methods Sigma（σ） values were calculated,according to imprecisions,biases （Bias） and allowable total error （TEa） by SYSMEX XE-2100D automatic hematological analyzer. The parameters included white blood cell （WBC） count,red blood cell （RBC） count,hemoglobin （Hb）,hematocrit （HCT）,platelet （PLT） count,neutrophil （NEUT）,lymphocyte （LYMPH）,monocyte （MONO）,eosinophil （EOS） and basophil （BASO）. Appropriate quality control rules were chosen with OPSpecs charts. Results The parameters achieving 6σ accounted for 80%,those achieving 5σ accounted for 10%,and the other 10% parameters did not achieve 5σ. For WBC count,RBC count,Hb,HCT,PLT count,Neut,LYMPH and BASO,quality control rule 1_3.5_s, N=3,R=1 should be used. For EOS,quality control rule 1₃_s, N=3,R=1 should be used. For MONO,quality control rule 1_2.5_s, N=3,R=1 should be used. These rules can meet 90% requirements for ensuring determination quality.Conclusions Clinical laboratories may choose appropriate quality control rules according to different parameters,in order to ensure determination quality.

Determinations of cardiac surgery-associated acute kidney injury

WU Dawei, NING Li, WEI Dianjun

2018, 33(1): 76-81. DOI: 10.3969/j.issn.1673-8640.2018.01.015

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：Cardiac surgery-associated acute kidney injury （CSA-AKI） is a common complication after cardiac surgery,which can usually develop chronic kidney disease,also resulting with high morbidity and mortality. Mortality,intensive care unit（ICU）residence time and hospitalization costs increase significantly in patients with CSA-AKI. Early diagnosis is important in the prevention and reduction of complications. The research of biomarkers of acute kidney injury in serum and urine is a focus. This review mainly focuses on pathogenesis,risk factors,classification and new early diagnosis biomarkers of CSA-AKI.

Development and comparison of Helicobacter pylori detection

CUI Junhua, LI Yihao, ZHOU Jiaye, WU Jiong, GUO Wei, PAN Baishen

2018, 33(1): 82-87. DOI: 10.3969/j.issn.1673-8640.2018.01.016

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：Many gastroduodenal diseases are associated with Helicobacter pylori （HP） infection,so accurate diagnosis of Hp infection is needed. Nowadays,several invasive and non-invasive tests are available for the detection of Hp. Endoscopy is used to collect specimens for invasive test,including endoscopic image,histology,culturing,rapid urease test and molecula. Methods . Non-invasive tests include urea breath test,stool antigen test and serological test. Some tests can analyze virulence and drug resistance as well. This review focuses on the development and application of Hp detection,in order to provide a reference for clinical application.

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