Table of Content

30 April 2020, Volume 35 Issue 4

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Orginal Article

Clinical evaluation of different detection methods of SARS-CoV-2 IgM and IgG antibodies in the COVID-19 diagnosis

ZHENG Peiming, CUI Facai, ZHANG Fuming, LI Gang

2020, 35(4):  291-294.  DOI: 10.3969/j.issn.1673-8640.2020.04.001

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Objective To compare different detection methods of severe acute respiratory syndrome coronavirus 2（SARS-CoV-2） IgM and IgG antibodies in the diagnosis of corona virus disease 2019（COVID-19）. Methodse Serum samples were collected from 25 patients diagnosed as COVID-19,and 27 patients without SARS-CoV-2 infection were enrolled as control group. SARS-CoV-2 IgM and IgG antibodies were detected by chemiluminescence and colloidal gold method. The procalcitonin（PCT）,C-reactive protein（CRP） and ferritin were also determined. Results The sensitivities of SARS-CoV-2 IgM and IgG antibodies by chemiluminescence were 48% and 56%,respectively,while those by colloidal gold method were 88% and 76%,respectively. The specificities were both 100% for the 2 methods. The total coincidence rates of IgM and IgG antibodies between the 2 methods were 68.9% and 73.3%,respectively. Serum PCT levels were increased in 36% COVID-19 patients,serum CRP levels were increased in 72% COVID-19 patients,and serum ferritin were increased in 84% COVID-19 patients. Conclusions The clinical performance of different detection methods of SARS-CoV-2 IgM and IgG antibodies are varied,which should be considered comprehensively in the clinical diagnosis of COVID-19.

Diagnostic roles of several parameters in corona virus disease 2019

CHEN Xing, OU Jingyi, HUANG Ying, TAN Mingkai, CHEN Jiabin, LIN Luping, LIANG Zhiwei, SHI Yaling

2020, 35(4):  295-299.  DOI: 10.3969/j.issn.1673-8640.2020.04.002

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Objective To observe the characteristics of blood routine items,biochemical indexes and D-dimer（DD） of general and severe corona virus disease 2019（COVID-19） patients and in the treatment of severe COVID-19,and to provide a reference for COVID-19 clinical diagnosis and treatment. Methodse Totally,296 COVID-19 patients were analyzed retrospectively. A total of 100 patients with common pneumonia were enrolled as control group. The changes of blood routine items,biochemical indexes and DD in 30 severe COVID-19 patients were analyzed dynamically. Results In severe COVID-19 group,lymphocyte percentage（LYMPH%）,the absolute value of lymphocyte（LYMPH#） and platelet（PLT） count were lower than those in general COVID-19 group（P<0.05）. The absolute value of neutrophil（NEUT#）,neutrophil/lymphocyte ratio（NLR）,aspartate aminotransferase（AST）,urea,creatine kinase（CK）,lactate dehydrogenase（LDH） and DD were higher than those in general COVID-19 group（P<0.05）. In severe COVID-19 group,white blood cell（WBC） count,PLT count,NEUT#,LYMPH#,LYMPH% were lower than those in control group,and NLR and LDH were higher（P<0.05）. Compared with control group,in general COVID-19 group,WBC count,PLT count,NEUT#,NLR,LYMPH#,urea,CK,DD and LDH were lower,and LYMPH% were higher（P<0.05）. There were 7 of 30 severe cases aggravated or turned to critical state,and their WBC count,NEUT# and NLR were higher than normal levels,while LYMPH# was lower than normal level. The above indexes in 23 patients who were improved or virus nucleic acid turned negative were gradually recovered to normal levels. Conclusions Blood routine items,biochemical indexes and DD are important for the diagnosis and treatment of COVID-19.

Correlation between neonatal hypoxic-ischemic encephalopathy and neurobehavior with S100B protein and neuron-specific enolase

FU Hui, XIONG Hong, KANG Wenqing, XU Bangli, LU Ruicun

2020, 35(4):  300-303.  DOI: 10.3969/j.issn.1673-8640.2020.04.003

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Objective To investigate the correlation between neonatal hypoxic-ischemic encephalopathy（HIE） and neurobehavior with S100B protein and neuron-specific enolase（NSE）. Methodse A total of 68 children with HIE were enrolled. They were classified into severe HIE group（20 cases）,moderate HIE group（25 cases） and mild HIE group（23 cases）. Totally,25 healthy newborns were enrolled as healthy control group. Serum S100B protein and NSE levels,neonatal behavioral neurological assessment（NBNA） score and NBNA score abnormality rate were determined. The changes of serum NSE and S100B protein in HIE group at 24 h,48 h,72 h and 7 d after birth were observed dynamically. Results In healthy control group,mild HIE group,moderate HIE group,severe HIE group,S100B protein and NSE levels and NBNA score abnormality rate were successively increased,and NBNA score was successively decreased（P<0.001）. Serum S100B protein and NSE levels were negatively correlated with NBNA score（r=-0.243 and -0.542,respectively,P<0.001）,and they were positively correlated with HIE severity（r=0.398 and 0.476,respectively,P<0.001）. With the extension of birth time,serum S100B protein and NSE levels in severe HIE group,moderate HIE group and mild HIE group were gradually decreased（P<0.001）. Serum S100B protein and NSE levels in mild HIE group,moderate HIE group and severe HIE group were successively increased at the same time point（P<0.001）. Conclusions Serum S100B protein and NSE levels are correlated with the severity of HIE and the neurobehavior of children,which can be used for the early diagnosis and prognosis of HIE.

Role of IDO/TTS ratio in the exclusion and disease assessment of RA

ZHOU Fujiang, WU Chengbin, GAO Shouxia, XU Chao, QIAN Wenying, HUANG Qiulan

2020, 35(4):  304-309.  DOI: 10.3969/j.issn.1673-8640.2020.04.004

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Objective To investigate the roles of indoleamine 2,3-dioxygenase（IDO）/tryptophanyl-tRNA-synthetase（TTS） ratio in rheumatoid arthritis（RA）. Methodse A total of 88 patients with RA（RA group） and 88 healthy subjects（healthy control group） were enrolled. Kynurenine（Kyn） and tryptophan（Trp） levels in plasma were determined by high performance liquid chromatography,and the Kyn/Trp ratio was calculated. Flow cytometry was used to determine the expressions of IDO and TTS in lymphocytes,and the IDO/TTS ratio was calculated. Fluorescence quantitative reverse transcription-polymerase chain reaction（RT-PCR） was used to determine peripheral blood mononuclear cells（PBMC）. The relative expressions of IDO mRNA and TTS mRNA were determined,and the ratio of IDO mRNA/TTS mRNA was calculated. According to the improved disease activity score 28（DAS28）,RA patients were classified into low activity group（DAS28≤3.2 points,35 cases）,medium activity group（DAS28>3.2-≤5.1 points,42 cases） and high activity levels（DAS28>5.1 points,11 cases）. Receiver operating characteristic（ROC） curve was used to evaluate the role of IDO/TTS ratio in the exclusion and disease assessment of RA. Results The levels of Kyn and Trp in plasma,the expression of TTS in lymphocytes and the relative expression of TTS mRNA in RA group were higher than those in healthy control group（P=0.000）,and the ratio of Kyn/Trp,the expression of IDO in lymphocytes,IDO/TTS ratio,the relative expression of IDO mRNA and the ratio of IDO mRNA/TTS mRNA were lower than those in healthy control group（P=0.000）. The IDO/TTS ratio of low activity group,medium activity group and high activity group decreased in order,and there was statistical significance among the groups（P<0.05）. ROC curve analysis showed that the area under curve（AUC） of IDO/TTS ratio in the exclusion of RA was 0.841,and the optimal cut-off value was 0.080,the sensitivity was 84.2%,and the specificity was 81.6%,the negative predictive value was 0.838,and the positive predictive value was 0.821. The AUC in the disease assessment of RA was 0.734,the optimal cut-off value was 0.042,the sensitivity was 72.1%,the specificity was 70.7%,the negative predictive value was 0.626,and the positive predictive value was 0.788. Conclusions The IDO/TTS ratio can be used to exclude RA,and also has a value for assessing RA.

Role of serum pro-gastrin releasing peptide in patients with small cell lung cancer

LI Huiping, ZHENG Dayong, YANG Shuai, ZHOU Yunli

2020, 35(4):  310-313.  DOI: 10.3969/j.issn.1673-8640.2020.04.005

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Objective To investigate the role of serum pro-gastrin-releasing peptide（Pro-GRP） in the diagnosis and treatment of small cell lung cancer（SCLC）. Methodse The levels of serum Pro-GRP,carcinoembryonic antigen（CEA）,squamous cell carcinoma antigen（SCC-Ag）,cytokeratin 19-fragment（CYFRA21-1） and neuron specific enolase（NSE） in 96 patients with SCLC（74 patients with limited SCLC and 22 patients with extensive SCLC）,63 patients with non-small cell lung cancer（NSCLC） and 76 healthy subjects（healthy control group） were determined. A total of 86 of 96 SCLC patients completed 1 cycle of chemotherapy. Results Compared with healthy control group and NSCLC group,the levels of Pro-GRP and NSE in SCLC group increased（P<0.001）,and serum SCC-Ag levels in SCLC group and NSCLC group were higher than that in healthy control group（P<0.001）. The levels of CEA and CYFRA21-1 were higher in NSCLC group than those in healthy control group and SCLC group（P<0.001）. The Pro-GRP level in patients with limited SCLC was lower than that in patients with extensive SCLC（P<0.01）. Pro-GRP and NSE were lower in SCLC patients after chemotherapy（P<0.001）. Receiver operating characteristic（ROC） curve showed that the sensitivity of Pro-GRP in diagnosing SCLC was 84.5%,and the specificity was 98.6%,which were higher than those of NSE in diagnosing SCLC（77.6%,79.5%,respectively）. The areas under curves（AUC） of Pro-GRP and NSE were 0.960 and 0.849,respectively. The optimal cut-off values were 76.31 ng/L and 13.87 ng/mL,respectively. Conclusions Serum Pro-GRP is of significance in the diagnosis,clinical staging and treatment monitoring of SCLC.

Role of fecal DNA level of Fusobacterium nucleatum and clbA+ Escherichia coli in the diagnosis of colorectal cancer

LIU Yan, LONG Yin, PAN Weijie, ZHANG Tong, WENG Wenhao, YU Ying

2020, 35(4):  314-317.  DOI: 10.3969/j.issn.1673-8640.2020.04.006

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Objective To investigate the role of fecal DNA level of Fusobacterium nucleatum and clbA+ Escherichia coli in the diagnosis of colorectal cancer. Methodse Real-time fluorescence quantitation polymerase chain reaction（PCR） was used to determine the DNA level of Fusobacterium nucleatum and clbA+Escherichia coli in 58 healthy subjects,38 adenoma patients and 115 colorectal cancer patients. Results The DNA levels of clbA+ Escherichia coli were 0.001 6（0.000 46-0.004 9） in healthy control group,0.002 6（0.000 45-0.008 8） in adenoma group and 0.006 3（0.002 0-0.036） in colorectal cancer group. Those of Fusobacterium nucleatum were 0.001 0（0.000 31-0.002 9）in healthy control group,0.003 3（0.001 1-0.011 0） in adenoma group and 0.006 4（0.002 9-0.012 0） in colorectal cancer group. The DNA level of Fusobacterium nucleatum and clbA+Escherichia coli in colorectal cancer group had statistical significance compared with healthy control group（P<0.000 1）. Receiver operating characteristic（ROC） curve analysis showed that the areas under curves（AUC）of clbA+ Escherichia coli and Fusobacterium nucleatum in the diagnosis of colorectal cancer were 0.712 and 0.750,respectively. The optimal cut-off values were 0.003 3 and 0.002 3,the sensitivities were 64.32% and 80.87%,and the specificities were 65.52% and 68.97%,respectively. The positive rates of clbA+ Escherichia coli and Fusobacterium nucleatum in the early stage of colorectal cancer were 75.7% and 80.5%,which were higher than those of carcinoembryonic antigen（CEA） and fecal occult blood test（FOBT）,and there was no statistical significance between early and advanced stages（P>0.05）. Conclusions The clbA+ Escherichia coli and Fusobacterium nucleatum are abundant in feces from colorectal cancer patients,which may be used as potential early determination biomarkers in colorectal cancer.

In vitro antibacterial activity and mechanism of artesunate on Propionibacterium acnes

CHEN Xiangming, HE Ping, LI Tin, ZHANG Haiqing, HU Yang

2020, 35(4):  318-322.  DOI: 10.3969/j.issn.1673-8640.2020.04.007

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Objective To investigate the in vitro antibacterial activity and mechanism of artesunate on Propionibacterium acnes. Methodse The minimum inhibitory concentrations（MIC） of artesunate combined with doxycycline hydrochloride to 14 standard and clinical isolates of Propionibacterium acnes were determined by broth microdilution method. Dynamic effects of the 2 drugs on the growth of Propionibacterium acnes were observed. Morphological changes of Propionibacterium acnes after artesunate treatment were observed by scanning electron microscopy. Results The MIC of artesunate was 0.750-3.000 mg/mL,and the MIC of doxycycline hydrochloride was 0.040-0.160 μg/mL. The 2 drugs showed additive or synergistic effect. The MIC difference between single drug and combined drugs was statistically significant（P<0.05）. Bacterial growth kinetics indicated that the combined drugs decreased the concentration of each drug,and the antibacterial action was rapid and long-lasting. Scanning electron microscopy showed that the bacteria became short after the action of artesunate,and the surface was wrinkled,sunk and curved. Conclusions Artesunate has antibacterial activity against Propionibacterium acnes. The combined action of artesunate and doxycycline hydrochloride is superior to single action. Artesunate has a morphological effect on Propionibacterium acnes.

Epidemiological characteristics of EB virus infection in inpatients from Hebei Children's Hospital

YAN Jianghong, JA Li, LI Wenhui, YANG Shuo, YAN Xiaotong, ZHAO Mengchuan, GUO Weiwei, LIU Yingye, LIU Zehao, WANG Le

2020, 35(4):  323-326.  DOI: 10.3969/j.issn.1673-8640.2020.04.008

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Objective To understand the epidemiological characteristics of Epstein-Barr virus（EBV） infection in inpatients from Hebei Children's Hospital,and to provide a reference for the diagnosis and prevention of EBV infection in children. Methodse The whole blood samples of children with EBV infection in Hebei Children's Hospital from January to December in 2017 were collected,and anti-viral capsid antigen（VCA） IgG and IgM antibodies,anti-early antigen（EA） antibody and anti-nuclear antigen-1（NA1） IgG antibody were determined by enzyme-linked immunosorbent assay（ELISA）. The antibody spectrum was analyzed. According to the determination results of 4 kinds of EBV antibodies,the primary infection group（anti-VCA-IgM positive,anti-NA1-IgG negative,anti-VCA-IgG positive or negative,anti-EA-IgG positive or negative）,sub-acute infection group （anti-VCA-IgG positive,anti-VCA-IgM positive or negative,anti-NA1-IgG positive or negative,anti-EA-IgG positive or negative）,previous infection group （anti-NA1-IgG positive,anti-VCA-IgG positive or negative,the rest negative） and uninfected group（all antibodies negative）. The positive rate of each group was analyzed according to age,season and sex. Results The antibody spectrums of 3 257 （73.17%） of 4 451 samples met the set requirements,indicating EBV infection,of which 380 cases were primary infection（8.54%）,616 cases of sub-acute infection（13.84%） and 2 261 cases of previous infection（50.80%）. There was statistical significance in the primary positive rate among different age groups（P<0.05）. The positive rate of pre-school age group（>3 years old） was the highest（P<0.05）. The positive rates of different months had statistical significance（P<0.05）,and the positive rate of EBV in July was higher than those in the other months（P<0.05）. There was no statistical significance in the EBV infection rate between females and males（P>0.05）. The disease spectrum of 380 cases of primary infection was mainly hematological diseases （infectious mononucleosis,acute agranulocytosis,thrombocytopenic purpura,EBV-associated hemophilia syndrome）,of which infectious mononucleosis was a common clinical disease;followed by respiratory diseases （acute bronchitis,herpes angina,acute tonsillitis）;the other diseases included neurological diseases and bloodstream infection,nephrotic syndrome and Kawasaki disease. Conclusions The positive rate of EBV in children in Hebei Children's Hospital varies with ages and months. Among the cases of primary infection,the main disease is hematological disease. The hospital should make corresponding preventive measures according to the epidemiological characteristics.

Relationship between four coagulation factors and C peptide and IR in T2DM patients with microangiopathy

ZUO Xuecan, CHU Yanling, LI Zhanping

2020, 35(4):  327-328.  DOI: 10.3969/j.issn.1673-8640.2020.04.009

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Objective To analyze the relationship between four coagulation factors [prothrombin time（PT）,activated partial thromboplastin time（APTT）,thrombin time（TT）and fibrinogen（Fib）] and C peptide and insulin resistance（IR） in type 2 diabetes mellitus（T2DM）patients with microangiopathy. Methodse A total of 100 T2DM patients with microangiopathy were enrolled as observation group,and 100 T2DM patients without microangiopathy were enrolled as control group. PT,APTT,TT,Fib,C peptide and IR levels were determined. Pearson correlation analysis was used to analyze the correlation between four coagulation factors and C peptide and IR levels. Results The levels of PT,APTT,TT and C peptide in observation group were lower than those in control group（P<0.001）,and the level of Fib and homeostasis mode assessment for insulin resistance（HOMA-IR） in observation group were higher than those in control group（P<0.001）. C peptide was positively correlated with PT and APTT（r=0.302 and 0.224,P<0.05）,and IR was negatively correlated with PT and APTT（r=-0.413 and -0.253,P<0.05）. Conclusions The coagulation dysfunction of T2DM patients with microangiopathy is related to the decreasing of C peptide and the increasing of IR.

Retrospective analysis of common drugs' therapeutic drug monitoring results in patients with mental diseases

YU Aiping, SUN Juanyu, XIE Haiyan, LU Wen, GUO Xiaoxia, SHI Yun

2020, 35(4):  330-333.  DOI: 10.3969/j.issn.1673-8640.2020.04.010

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Objective To study the role of therapeutic drug monitoring（TDM） by the retrospective analysis of 5 common drugs' TDM results in patients with mental diseases. Methodse The TDM results of 2 mood stabilizers（valproic acid,carbamazepine） and 3 antipsychotics（clozapine,olanzapine,palisperidone） in 721 patients with mental diseases were analyzed retrospectively,and the control rates of the 5 drugs before and after TDM were compared. Results The control rates of valproic acid and carbamazepine before TDM were 31.15% and 33.33%,while those after TDM increased to 81.53% and 81.82%,respectively. The control rates of clozapine,olanzapine and palisperidone before TDM were 24.35%,67.32% and 68.75%,while those after TDM increased to 62.61%,83.01% and 83.13%,respectively. Compared with before TDM,the control rates of the 5 drugs after TDM were increased（P<0.05）. After TDM,the increase rate of valproic acid for the control rate（50.38%） was higher than those of clozapine,olanzapine and palisperidone（38.26%,15.69% and 14.38%,respectively）（P<0.05）,while the increase rate of carbamazepine for the control rate（48.49%） was higher than those of olanzapine and palisperidone（15.69% and 14.38%,respectively）（P<0.05）. Conclusions TDM can effectively guide clinical drug use,realize individual drug use and achieve optimal therapeutic effect.

Analysis on the results of coagulation four parameters and D-dimer in healthy pregnant women in Lan

zhou YE Ping, DONG Xuemei, ZHANG Chong, DU Xiaozhong

2020, 35(4):  334-337.  DOI: 10.3969/j.issn.1673-8640.2020.04.011

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Objective To analyze the results of coagulation four parameters and D-dimer（DD） in healthy pregnant women with different gestational weeks in Lanzhou,and to provide a reference for assessing the status of maternal coagulation. Methodse Retrospective analysis was performed in 7 802 pregnant women and 168 non-pregnant women. According to different gestational weeks,7 802 pregnant women were classified into early pregnancy group,middle pregnancy group and late pregnancy group. Prothrombin time（PT）,activated partial thromboplastin time（APTT）,thrombin time（TT）,fibrinogen（Fib） and DD were determined. Results With the progress of gestational weeks,PT,APTT and TT in pregnant women were gradually shortened,and Fib and DD were gradually increased. Compared with pregnant groups,Fib and DD had statistical significance in non-pregnant group（P<0.01）. Conclusions The reference of PT,APTT,TT,Fib and DD for each pregnancy stage in Lanzhou has been established,which improves the application of maternal coagulation function determinations in clinical diagnosis.

Analysis of pediatric urinary stone composition among Uygur children in Hetian MA Jing,

WANG Changmin, HUANG Guohong, XIA Zhigang, LI Shuai, LIU Hong

2020, 35(4):  338-341.  DOI: 10.3969/j.issn.1673-8640.2020.04.012

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Objective To study pediatric urinary stone composition among Uygur children in Hetian,and to provide a reference for the treatment of pediatric urinary stone and the prevention of recurrence. Methodse A total of 203 cases of pediatric urinary stones from Uygur children in Hetian were collected,and the composition was analyzed by infrared spectroscopy. Results Among the 203 cases,144 cases were from males（70.94%）,and 59 cases were from females（29.06%）. The peak age of onset was preschool period,accounting for 51.72%. Totally,123 cases came from kidney（60.59%）,37 cases came from ureter（18.23%）,33 cases came from bladder（16.26%）,and 10 cases came from urethra（4.93%）. Among 39 cases（19.21%） of single component,calcium oxalate monohydrate was the main component,accounting for 48.72%（19 cases）,followed by ammonium urate accounting for 35.90%（14 cases）. There was no statistical significance in the composition of single component between males and females（χ²=3.186,P>0.05）. For mixed components,calcium oxalate dihydrate and ammonium urate were the main components,accounting for 34.15%（56 cases）,followed by calcium oxalate monohydrate and calcium oxalate dihydrate,accounting for 16.46%（27 cases）. There was statistical significance in the composition of mixed components between males and females（χ²=53.081,P<0.01）. Conclusions Calcium oxalate is the main kind of pediatric urinary stone composition among Uygur children in Hetian,and the difference exists between different ages and sex.

Application of vaginal microbial chip detection technique in the detection of vaginal secretion Candida

CHEN Leiming, CHEN Yisheng, LI Cui

2020, 35(4):  346-348.  DOI: 10.3969/j.issn.1673-8640.2020.04.014

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Objective To analyze the role of vaginal microbial chip detection technique in the detection of vaginal secretion Candida. Methodse A total of 233 samples of vaginal secretion were collected. Routine microscopy,fungal culturing and vaginal microbial chip detection technique were performed. The results were analyzed statistically. Results The results of fungal culturing were used as gold standard,and the Kappa values were 0.415 and 0.821 by routine microscopy and vaginal microbial chip detection technique,respectively,and the areas under receiver operating characteristic（ROC） curves were 0.73 and 0.91,respectively. Conclusions The vaginal microbial chip detection technique is easy to operate,improves the efficiency of Candida detection in vaginal secretion samples,and has accuracy. It plays a role in timely and accurate diagnosis of Candida vaginitis.

Roles of automated image analysis system and CytoDiff flow cytometry for the diagnosis and treatment of leucopenia

GUO Ping, CHEN Liting, LIAO Bing, WANG Jianbiao

2020, 35(4):  349-353.  DOI: 10.3969/j.issn.1673-8640.2020.04.015

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Objective To evaluate the roles of automated image analysis system and CytoDiff flow cytometry for the diagnosis and treatment of leucopenia. Methodse A total of 181 patients with white blood cell count（0.5-2.0）×10⁹/L were enrolled and determined for differentiation and count by automated image analysis system and CytoDiff flow cytometry. Time,precision,correlation of blasts,sensitivity and specificity were compared with microscopy（standard method）. Results The median times were 864,277,190 and 364 s by microscopy,CytoDiff flow cytometry,DM96 preclassification and DM96 reclassification,respectively. The variation of CytoDiff flow cytometry for neutrophils,lymphocytes,monocytes and blasts was better than those of the other 3 methods. The correlation coefficients（r） between CytoDiff flow cytometry,DM96 preclassification,DM96 reclassification and microscopy were 0.937,0.507 and 0.990,respectively. The sensitivity and specificity of CytoDiff flow cytometry were 94.90% and 98.80%,respectively. The sensitivities of DM96 preclassification and DM96 reclassification were 54.08% and 79.59%,and the specificities were 74.69% and 100.00%,respectively. Conclusions DM96 reclassification and CytoDiff flow cytometry can detect blasts in leucopenia samples with high efficiency and accuracy and meet the demand of clinic for the curative effect monitoring and prognostic analysis.

Establishment of a digital PCR mutation detection platform for ctDNA in patients with intrahepatic cholangiocarcinoma and its clinical application

DAI Qian, HUANG Fei, WANG Yupeng, HUANG Ao, CHENG Jianwen, PAN Baishen, GUO Wei, ZHOU Jian, FAN Jia, YANG Xinrong, WANG Beili

2020, 35(4):  354-362.  DOI: 10.3969/j.issn.1673-8640.2020.04.016

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Objective To establish a digital polymerase chain reaction（dPCR） detection platform for KRAS G12D,TP53 C242S and IDH1 R132C mutations,and to preliminarily evaluate its detection performance and clinical application value. Methodse A total of 22 intrahepatic cholangiocarcinoma（ICC） patients who underwent resection were enrolled. The primers and probes of KRAS G12D,TP53 C242S and IDH1 R132C mutation sites were designed to establish a dPCR mutation detection platform. The accuracy,precision,detection limit of blank,functional sensitivity and linear range of the platform were validated according to self-made standard quality particles with different concentrations. The results of dPCR in Oseq-ctDNA in peripheral blood and Oseq-T targeted sequencing in tissues were compared. Peripheral blood samples were collected and followed up every 6 months after resection to evaluate the role of the platform in monitoring the efficiency and prognosis of ICC patients. Results The accuracy of dPCR platform was good,and the deviation among the 3 abundances of the 3 mutations（KRAS G12D,TP53 C242S and IDH1 R132C） and the theoretical value was less than ±15%. The detection limit of blank was 4 copies,the functional sensitivity was 0.1%,and the linearity was good in the range of 0.1%-10%. In 22 ICC patients,the sensitivity,specificity and area under curve（AUC） of ctDNA were 31.8%,100% and 0.659,those for carbohydrate antigen 19-9（CA19-9） combined with ctDNA were 68.2%,100% and 0.841. The consistency of dPCR and Oseq-ctDNA in peripheral blood was high（kappa=0.792,P=0.007）. The results of follow-up showed that the copy number of KRAS G12D mutation was increased in 1 ICC patient after resection for 18 months,which were the same as the recurrence time confirmed by imaging. Conclusions A dPCR detection platform for detecting KRAS G12D,TP53 C242S and IDH1 R132C mutations has been established,which can be used in the diagnosis,efficiency evaluation and dynamic monitoring of ICC patients after resection.

TNF and CRT dual signaling for promoting NLRP3 inflammasome activation in synoviocytes for RA patients

LIU Yixin, WEI Wei, WANG Yang, BAI Yingyu, WAN Chunyou, MA Jun, ZHENG Fang

2020, 35(4):  363-369.  DOI: 10.3969/j.issn.1673-8640.2020.04.017

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Objective To investigate the effect of tumor necrosis factor-alpha（TNF-α） and calreticulin（CRT） dual signaling on nucleotide-binding oligomerization domain-like receptor protein 3（NLRP3） inflammasome activation in fibroblast-like synoviocytes（FLS） of rheumatoid arthritis（RA）. Methodse Indirect immunofluorescence staining was used to determine the expressions of NLRP3 and apoptosis-associated speck-like protein（ASC） in synovial tissue of 12 RA and 10 osteoarthritis（OA） patients,and co-localizations with podoplanin and CD248 were performed. RA FLS in synovial tissue of RA patients was isolated by collagenase digestion and cultured in vitro. The cells were treated with different concentrations of TNF-α or lipopolysaccharide（LPS）（stimulant）,and the protein and mRNA expressions of NLRP3,pro-interleukin 1 beta（pro-IL-1β） and pro-interleukin 18（pro-IL-18） were determined by western blot and real-time fluorescence quantitative polymerase chain reaction（qRT-PCR）,respectively. The supernatant of cells was discarded,and then the cells were treated with Nigericin or CRT. The expression of cysteine containing aspartate specific protease-1（Caspase-1） activated fragment p20 in FLS was determined by western blot,and the cell culture supernatant was collected for secretive interleukin（IL）-1β and IL-18 determinations by enzyme-linked immunosorbent assay（ELISA）. Results The expression of NLRP3 and ASC in RA synovial tissue was higher than that in OA（P<0.01）. The co-localizations of NLRP3,ASC and cleaved IL-1β with podoplanin and CD248 were observed. In in vitro experiments,TNF-α could promote the protein expressions of NLRP3 and pro-IL-1β,and the mRNA expressions were higher than those of control group（treated without stimulant）（P<0.05,P<0.001）,whereas LPS had no effect on the priming of NLRP3 inflammasome in RA FLS. TNF-α/Nigericin or TNF-α/CRT dual signaling can promote the activation of Caspase-1 in FLS,represented by the increasing of Caspase-1 activated fragment p20 in a dose-dependent manner,further leading to the increasing of secretive IL-1β compared with that in control group（P<0.05）. Conclusions TNF-α/CRT dual signaling promotes the activation of NLRP3 inflammasome in FLS of RA.

Standards of CLSI clinical laboratory automation systems

FU Yawen, DU Yuzhen, GAO Feng

2020, 35(4):  370-373.  DOI: 10.3969/j.issn.1673-8640.2020.04.018

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Clinical laboratory automation system can improve the management level of laboratories,improve work efficiency,reduce error rate,reduce the amount of blood used,shorten sample turnaround time,and improve the bio-safety of clinical laboratories. The construction of efficient clinical laboratory automation system needs technical guidelines,but there is no relevant guiding standards in China. This review introduces several approved standards on clinical laboratory automation systems issued by the Clinical and Laboratory Standards Institute（CLSI）,which provides a reference for the construction of clinical laboratory automation system.

Progressive research of Alzheimer's disease-related blood markers

CHEN Xiaotong, LIN Yong

2020, 35(4):  374-379.  DOI: 10.3969/j.issn.1673-8640.2020.04.019

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Alzheimer's disease（AD）is a degenerative disease of the central nervous system with clinical manifestations of memory impairment,cognitive impairment,later personality and behavioral changes. Recently,studies have shown that many blood markers are closely related to AD,like protein,gene mutation,microRNA（miRNA）,which may play a certain role in predicting the risk of AD,reflecting the stage of AD and supporting the diagnosis of AD. In addition,the determination method of blood markers is simple and easy,therefore screening ideal AD-related blood markers is beneficial to the screening and treatment of AD. In this review,the latest research status of AD-related blood markers is summarized,and the possibility and limitation of its clinical application are discussed.

Research progress of myeloid differentiation factor 88 polymorphism

HU Yuyi, CHEN Pu, GUO Wei, PAN Baishen

2020, 35(4):  380-386.  DOI: 10.3969/j.issn.1673-8640.2020.04.020

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Myeloid differentiation factor 88（MYD88）is an important adaptor protein mediating the signal transduction of Toll-like receptors（TLR）,interleukin 1 receptor（IL-1R）and interleukin 18 receptor（IL-18R）,which plays a key role in innate immunity. MYD88-dependent pathways play roles in the pathogenesis of multiple pathogens and have relations with tumors,infectious diseases,autoimmune diseases and so on,which are considered to be important targets for the intervention of these diseases. The L265P mutant in MYD88,an amino acid change（L265P）in the MYD88 Toll-like receptor/interleukin 1 receptor（TIR）,promotes Janus kinase-signal transducer and activator of transcription 3（JAK-STAT3） signaling pathway by enhancing transcription factors such as nuclear factor kappa B（NF-κB）,and mediates the production of inflammatory factors such as interleukin（IL）-6,IL-10 and interferon-beta（IFN-β）. The structure and fundamental function of MYD88 and its role in signal transduction are reviewed. Furthermore,the association between MYD88 L265P and diseases are also illustrated.