Table of Content

28 February 2021, Volume 36 Issue 2

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Thinking and prospect in the diagnosis and treatment of rare diseases

YU Tingting, FU Qihua

2021, 36(2):  119-121.  DOI: 10.3969/j.issn.1673-8640.2021.02.001

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With the rapid development of medical technology,great progress has been made in the diagnosis and treatment of rare diseases in recent years. High-throughput sequencing technology plays an important role in the clinical diagnosis of rare diseases. Through gene diagnosis,clinicians have a new understanding on the mutations and clinical phenotypes of rare diseases. And targeted treatment strategies and prognosis management were adopted. In combination with several reports in this special issue of rare diseases,we reviewed the phenotype analysis of rare diseases,the reasonable application of detection techniques,the accurate analysis of genetic results,and the development of orphan drugs. It is hoped that it will provide reference for clinical practice of rare diseases.

Progress in laboratory diagnosis of rare diseases

ZHANG Yi, WANG Zengge, WANG Jian

2021, 36(2):  122-129.  DOI: 10.3969/j.issn.1673-8640.2021.02.002

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Rare diseases are a series of single disease with a low incidence severe condition,and rapid progress. Early diagnosis can effectively delay the course of disease and improve the quality of life of patients. With the popularization of high-throughput sequencing,gradual improvement of third-generation sequencing technique and advances in multiomics research technique,laboratory diagnostic methods for rare diseases have continued to diversify and become more efficient. The integration of high-speed updated big data,selective combination of cytogenetics and molecular genetics methods,and flexible use of biochemical detection indicators will effectively support and promote the diagnosis of rare diseases. Technological progress has greatly improved the diagnostic level of rare diseases,and a comprehensive understanding of laboratory diagnostic techniques for rare diseases will help to give full play to its advantages in clinical applications. In this article,we reviewed the existing laboratory diagnostic techniques for rare diseases and their progress in order to improve clinical understanding of different diagnostic techniques and help to choose suitable testing methods.

Analysis of clinical features and genetic variation of Sotos syndrome

ZHENG Hongxue, CHEN Yao, YIN Liping, LI Xin, DING Yu, LI Juan, WANG Xiumin

2021, 36(2):  130-134.  DOI: 10.3969/j.issn.1673-8640.2021.02.003

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Objective To analyze clinical characteristics and genetic variation of a case of Sotos syndrome misdiagnosed as precocious puberty. Methods The clinical data and related laboratory test results from one child with Sotos syndrome misdiagnosed as precocious puberty were retrospectively analyzed . Results Clinical manifestations of the child presented overgrowth,developmental delay,and typical facial appearance（macrocephaly,broad forehead,pointed chin,high palate）. The patient was misdiagnosed as precocious puberty in other hospital and treated with triptorelin acetate for 15 months,but growth rate has not slowed down. Heterozygous missense variants in nuclear receptor-binding SET-domain-containing protein 1（NSD1）gene was identified in proband by gene sequencing,which was c.5854C>T（p.Arg1952Trp）. His father had the same heterozygous mutation. This mutation had been classified to likely pathogenic mutation by American College of Medical Genetics and Genomics（ACMG） variation classification criteria. Conclusion The diagnosis of Sotos syndrome is confirmed in this child and NSD1 gene mutation is the cause. Sotos syndrome is characterized by overgrowth and bone age advanced. The results of the provocation test cannot be distinguished from precocious puberty alone. The clinical development of secondary sexual characteristics should be strictly evaluated to avoid misdiagnosis.

Analysis of clinical manifestations and genetic characteristics of autosomal recessive cutis laxa caused by PYCR1 variants

WU Wenyong, SHANGGUAN Huakun, CHEN Ruimin

2021, 36(2):  135-139.  DOI: 10.3969/j.issn.1673-8640.2021.02.004

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Objective To analyze the clinical manifestations and genetic characteristics of autosomal recessive cutis laxa（ARCL） caused by PYCR1 variants. Methods The clinical data of a patient with ARCL caused by PYCR1 variants were collected. The patient and his parents' blood samples were taken for whole exome sequencing. Clinical manifestations and genetic characteristics of ARCL were analyzed in combination with the literatures. Results The patient with a height of 81.7cm（-2.48s）and a weight of 8.0 kg（-2.68s）. Physical examination showed special facial features,torticollis,exposed thoracic and abdominal wall veins,obvious loose and wrinkled body skin. Previous history showed cryptorchidism and indirect inguinal hernia. WES identified compound heterozygous variants（c.345delC/p.Arg115Glyfs*7 and c.413G>A/p.Gly138Asp）of exon 4 in PYCR1 gene,among which c.413G>A/p.Gly138Asp was a new variant unreported. Combined with previously published data,115 cases of PYCR1-related ARCL mainly presented with skin laxity [100%（84/84）],obvious skin wrinkles [97.8%（88/90）],mental retardation or global developmental disabilities [95.2%（100/105）],joint relaxation [94.2%（65/69）],decreased bone density [73.8%（45/61）],clasped thumb [63.4%（26/41）],dislocation of hip joint [59.6%（53/89）],triangular face [93.1%（27/29）],microcephaly [72.6%（45/62）] and short stature [83.6%（51/61）]. Conclusion Patients presented with presenile appearance,mental retardation,global developmental disabilities,special facial features,short stature and joint relaxation should be considered for PYCR1-related ARCL. Genetic test aids to confirm diagnosis.

Analysis of clinical and genetic characteristics of GRIN1 mutation-related underdevelopment

LU Yaya, DING Yu, YAO Ruen, WANG Yirou, ZHANG Qianwen, LI Qun, WANG Jian, WANG Xiumin, LOU Dan

2021, 36(2):  140-146.  DOI: 10.3969/j.issn.1673-8640.2021.02.005

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Objective To analyze clinical and genetic characteristics of GRIN1 mutation-related underdevelopment. Methods Clinical data of 2 developmental retardation children were collected,and targeted gene sequencing（TGS） was performed in the 2 children and their parents. The clinical and genetic characteristics of developmental retardation children were analyzed together with literature review. Results The 2 children showed mental retardation,language backwardness,motor backwardness,no special face or epilepsy. One of them was complicated with dwarfism. The results of TGS showed that there were mutations in GRIN1 gene of the 2 children. One had missense variant c.1672t > G,p.Phe558Val（heterozygous）,and the other had missense variant c.1852g > A,p.Gly618Ser（heterozygous）. Both of their parents were normal at the same gene loci. Conclusion GRIN1 genetic mutation on the developmental retardation children is newly discovered. TGS is helpful to make clear the molecular mechanism of developmental retardation.

Analysis of 4 cases of acrodysostosis type 1 caused by PRKAR1A gene mutation

CHEN Fei, QIN Zailong, CHEN Shaoke, FAN Xin, LI Chuan, YI Shang, SHEN Yiping, LUO Jingsi

2021, 36(2):  147-152.  DOI: 10.3969/j.issn.1673-8640.2021.02.006

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Objective To study 4 cases of acrodysostosis 1 caused by PRKAR1A gene mutation based on their clinical manifestations and laboratory parameters. Methods Clinical data and laboratory testing results of 4 cases of acrodysostosis 1 were collected,and whole exome high-throughput sequencing was performed. Phenotype-driven variant filters were performed to identify candidate variants,and Sanger sequencing was used to verify the parental origin of the variants. The pathogenicity of genetic variation was classified according to Standards and Guidelines for the Interpretation of Sequence Variants. Results The main clinical manifestations of the 4 cases were short stature（-3s - -6s）,low weight（-2s - -4s）,brachydactyly,dysmorphic facial features and multi-hormone resistance. Different clinical manifestations were observed among individuals,and some patients had partial growth hormone deficiency. Pathogenic mutations in PRKAR1A were detected in the 4 cases. Of them,3 cases had the mutation c.1102C>T/p.Arg368*,and the one had the mutation c.1118A>G/p.Tyr373Cys. Conclusion Totally,2 new mutations in PRKAR1A have been detected,and there is heterogeneity in the clinical manifestations of patients with acrodysostosis 1.

Analysis on changes of gut microbiota in children with autism spectrum disorder based on high-throughput sequencing

ZENG Peipei, ZENG Ting, DENG Liangqiong, GUO Hao, FENG Yushan, HUANG Liping, LI Honghui

2021, 36(2):  153-161.  DOI: 10.3969/j.issn.1673-8640.2021.02.007

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Objective To investigate the changes of gut microbiota in children with autism spectrum disorders（ASD）. Methods A total of 35 children with ASD were enrolled into ASD group,and 35 healthy children were assigned to healthy control group. Genomic DNA was extracted from fecal samples,and Illumina sequencing platform was used for sequencing analysis to determine the characteristics of gut microbiota. Results There was difference in gut microbiota between ASD and healthy control groups. At the phylum level,there was no significant difference in the relative abundance of the Firmicutes,Bacteroidetes,Proteobacteria or Actinobacteria（P>0.05）. There was a significant difference in the structure of Candidatus Saccharibacteria between the 2 groups（P=0.01）,but the relative abundance in the structure of gut microbiota was very low. At the genus level,there were significant differences in the relative abundance of Subdoligranulum,Holdemania,Candidatus Saccharibacteria noname,Prevotella,Burkholderiales noname,Gordonibacter,Alistipes,Solobacterium,Parasutterella,Anaerotruncus,Aggregatibacter,Comamonas,Gemella and Granulicatella between the 2 groups（P<0.05）. Further analysis of gut microbiota showed that the relative abundance of Alistipes onderdonkii,Alistipes putredinis,Bacteroides finegoldii,Alistipes shahii,Streptococcus vestibularis,Holdemania filiformis,Bacteroides massiliensis,Burkholderiales bacterium-1-1-47,Megamonas funiformis,Parabacteroides merdae,Ruminococcus flavefaciens,Lachnospiraceae bacterium_3_1_57FAA_CT1 and Parasutterella excrementihominis in ASD group were higher than those in healthy control group,but the relative abundance of Granulicatella adiacens,Fusobacterium mortiferum,Solobacterium moorei,Eubacterium rectale,Candidate division_TM7_single_cell_isolate_TM7c and Gemella morbillorum in the ASD froup were lower than those in the healthy control group（P<0.05）. Conclusion Intestinal microecological disorders exist in ASD children,but ASD children do not have a specific intestinal microbial composition.

Clinical value of combined detection of serum HA and HAS2 in diagnosis and therapeutic monitoring of bone tumors

SHENG Yumeng, XU Jing, LIU Yiwen, HE Yiqing, LIU Hua, HUANG Yi, HU Jiajie, CUI Lian, GAO Feng, YANG Cuixia

2021, 36(2):  162-166.  DOI: 10.3969/j.issn.1673-8640.2021.02.008

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Objective To investigate the application of serum hyaluronan（HA） and hyaluronan synthetase 2（HAS2） in bone tumor. Methods A total of 35 patients with bone tumor,40 patients with bone infection and 60 healthy subjects were enrolled. The expression levels of HA,HAS2 and HAS2 mRNA were determined. Receiver operating characteristic（ROC）curve was used to analyze the diagnostic efficiency of each marker. Results The expression levels of HA,HAS2 and HAS2 mRNA in patients with bone tumor before and after surgery were significantly higher than those in healthy subjects（P<0.05）. The expression levels of HAS2 and HAS2 mRNA in patients with bone tumor before and after surgery were significantly higher than those in patients with bone infection（P<0.05）,but there was no significant difference in serum HA level between them（P>0.05）. After bone tumors were removed,the expression levels of HA,HAS2 and HAS2 mRNA were significantly reduced（P<0.05）. ROC curve analysis showed that the area under the curve（AUC） of HA,Has2 and Has2 mRNA single detection and combined detection were 0.888,0.651,0.747 and 0.956,respectively,when healthy subjects were taken as control;and the AUC were 0.485,0.782,0.630 and 0.798,respectively,when bone infection group was taken as control. Conclusions The combined determination of HA ,HAS2 and HAS2 mRNA plays a role in the auxiliary diagnosis and therapeutic monitoring of bone tumor.

Inflammatory cytokine levels in patients with philadelphia chromosome-negative myeloproliferative neoplasms

CHEN Pu, MA Yangting, CHEN Nan, YU Zhenglin, CHENG Yunfeng, PAN Boshen, GUO Wei, WANG Beili

2021, 36(2):  167-172.  DOI: 10.3969/j.issn.1673-8640.2021.02.009

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Objective To investigate serum levels of inflammatory cytokines in patients with classical myeloproliferative neoplasms（MPN） [tumor necrosis factor-alpha（TNF-α）,interleukin-1 beta（IL-1β）,interleukin-2 receptor（IL-2R）,interleukin-6（IL-6）,interleukin-8（IL-8）,interleukin-10（IL-10）] and their clinical significance. Methods The serum levels of TNF-α,IL-1β,IL-2R,IL-6,IL-8 and IL-10 were detected by chemiluminescence immunoassay（CLIA）in 50 patients with Philadelphia chromosome-negative（Ph^-） newly diagnosed MPN [MPN group,including 16 cases of polycythemia vera（PV）,25 cases of essential thrombocytosis（ET） and 9 cases of primary myelofibrosis（PMF）] and 20 healthy volunteers（control group）. Sanger sequencing was used to detect JAK2 and MPL mutations,and CALR mutation was detected by real-time fluorescent polymerase chain reaction（PCR） in MPN patients. Results The serum levels of TNF-α,IL-2R,IL-6 and IL-8 in MPN group were significantly higher than those in control group（P<0.001）,while IL-10 level was on the contrary（P<0.001）. There was no significant difference in serum IL-1β level between the two groups（P>0.05）. IL-2R level in PMF group was higher than that in PV and ET groups（P<0.05）. Among the JAK2 V617F mutation-positive patients,IL-2R level in the PMF group was also significantly higher than that in the PV and ET groups（P<0.01）;the levels of TNF-α and IL-8 in the PMF group were higher than those in the ET group（P<0.05）;the level of IL-6 in the PMF group was higher than that of the PV group（P<0.05）. Interestingly,the level of TNF-α in MPN patients with marrow fibrosis（MF）-3 was higher than that in patients with MF-0（P<0.05） and its IL-2R level was higher than that of patients with MF-2（P<0.05）. Conclusion Inflammatory cytokines（TNF-α,IL-1β,IL-2R,IL-6,IL-8 and IL-10）are abnormally expressed in MPN patients,and there are differences in the expression among the three subtypes of MPN. Cytokine levels might become potential reference indicator for MPN diagnostic classification and follow-up.

Comparison of application of 4 screening models for thalassemia

ZHUANG Qianmei, LIU Xiaolong, HUANG Hailong, WANG Yuanbai, ZHUANG Jianlong, XU Liangpu, HONG Guolin

2021, 36(2):  173-176.  DOI: 10.3969/j.issn.1673-8640.2021.02.010

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Objective To evaluate the effects of 4 common screening models for thalassemia. Methods A total of 670 couples carrying thalassaemia gene and 568 couples without thalassaemia gene were enrolled in this study. The genetic diagnosis results for thalassemia were used as golden standard. Routine blood analysis and hemoglobin（Hb） electrophoresis analysis were performed. Results The positive predictive values of Mode 1,Mode 2,Mode 3 and Mode 4 were 7.39%,7.25%,7.86% and 6.76%;its negative predictive values were 94.66%,98.52%,93.90% and 96.84%;the specificities were 53.53%,17.24%,88.88% and 13.19%;the sensitivities were 55.13%,96.15%,14.10% and 93.59%;and the missed diagnosis rates were 44.87%,3.85%,85.90% and 6.41%,respectively. Mode 2 had the highest negative predictive value and the lowest rate of missed diagnosis. Conclusion Mode 2 is the optimal screening mode for thalassemia. It should be selected for thalassemia screening as much as possible.

Efficacy evaluation and application value of screening carbapenemase in enterobacteriaceae by mCIM and eCIM

YIN Juan, WANG Yingchao, SUI Yang, JIANG Chun, SHI Yunqi, ZHU Chaowang

2021, 36(2):  177-180.  DOI: 10.3969/j.issn.1673-8640.2021.02.011

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Objective To investigate the capacity and application value of modified carbapenem inactivation method（mCIM）and ethylene diamine tetraacetic acid-carbapenem inactivation method（eCIM）for phenotypic detection of carbapenemase-producing Enterobacteriaceae. Methods A total of 117 strains of carbapenem-resistant Enterobacteriaceae（CRE） （experimental group）and 50 strains of carbapenem-sensitive Enterobacteriaceae （control group）were collected. Phenotypic screening was performed by modified Hodge test（MHT）,mCIM and eCIM in both groups,respectively. Common carbapenem-resistant genes were detected by polymerase chain reaction（PCR）. The consistency of phenotypic screening and gene examinations was analyzed statistically. Results When the detection result of carbapenem-resistant genes was taken as golden standard,the sensitivity of MHT for screening carbapenemase was 87.0%,and the specificity was 100.0%;both sensitivity and specificity of mCIM were 100.0%;the sensitivity and specificity of eCIM were 82.4% and 100.0% respectively. Conclusion mCIM is more sensitive than MHT in screening carbapenemase. The combined detection of mCIM and eCIM can effectively detect carbapenemase and distinguish the type of carbapenemase,which can play a guiding role in clinical medication.

The correlation between serum IL-17 and IL-35 and vitamin D in Hashimoto thyroiditis

KE Wencai, GU Yunxia, LIU Jie, JIN Xiaoling

2021, 36(2):  181-184.  DOI: 10.3969/j.issn.1673-8640.2021.02.012

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Objective To investigate the correlation between serum interleukin（IL）-17 and IL-35 and vitamin D in Hashimoto thyroiditis. Methods A total of 152 HT patients（HT group） were selected and divided into normal thyroid function group（51 cases）,subclinical hypothyroidism group（50 cases） and clinical hypothyroidism group（51 cases） according to the thyroid hormone level. Fifty healthy persons were taken as normal control. Serum levels of free triiodothyronine（FT₃）,free thyroxine（FT₄）,thyroid stimulating hormone（TSH）,thyroid globulin antibody（TgAb）,thyroid peroxidase antibody（TPOAb）,25-hydroxyvitamin D[25（OH） D],IL-17 and IL-35 were detected in all subjects. Pearson correlation analysis was used to evaluate the correlation between various indicators. Results Serum 25（OH）D and IL-35 levels in HT group were significantly lower than those in the normal control group（P<0.05）,while IL-17 level was significantly higher than that in the normal control group（P<0.05）.Compared with the normal thyroid function group,the serum 25（OH）D level of the clinical hypothyroidism group was significantly reduced（P<0.05）,and the vitamin D deficiency rate of the subclinical hypothyroidism group and clinical hypothyroidism group was increased（P<0.05）.There was no significant difference in serum 25（OH）D between subclinical hypothyroidism group and clinical hypothyroidism group（P>0.05）. Pearson correlation analysis showed that after adjustment of age,serum 25（OH）D level in the HT group was negatively correlated with TSH,TPOAb,and IL-17（r values were -0.320,-0.254,and -0.339,P<0.05）,positively correlated with IL-35（r=0.243,P<0.05）,but not related with FT₃,FT₄,or TgAb（r values were 0.181,0.012,0.018,P>0.05）. Conclusion The serum vitamin D level of HT patients is obviously insufficient,which is correlated with IL-17 and IL-35,suggesting that vitamin D level may be related to the onset and severity of HT.

Analysis of unknown source mutation in STR loci in paternity testing

LAN Feifei, DING Hongke, CHEN Yanbing, YU Lihua, YIN Aihua

2021, 36(2):  185-189.  DOI: 10.3969/j.issn.1673-8640.2021.02.013

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Objective To investigate the phenomenon,reason and parentage index calculation of unsure mutation source in STR loci in paternity testing. Methods Chelex-100 method was used to extract DNA from 4 385 family samples. The allele genotyping of STR loci was analyzed by capillary electrophoresis after amplification with polymerase chain reaction（PCR）. Mutated STR loci were verified by Identifiler system or Microreader 23sp ID system,respectively,and then validated by locus-specific primer amplification and sequencing. Results Mutations of unsure source were found in D12S391,TPOX and D8S1179 loci in two families. The authenticity of the mutation occurrence was confirmed by different detection systems. Sequencing results showed that the mutations of D12S391 and TPOX loci came from their mother,and both were one-step mutations. Conclusion Mutations of unknown source of STR locus has great influence on the calculation of the parentage indexand the identification conclusion. For mutations of unknown source,primers should be designed for amplification and sequencing to verify the source. The parentage index should be calculated correctly to ensure the accuracy and scientificity of the identification conclusion.

Diagnostic role of NLR,CRP,PCT,DD and NT-proBNP in severe corona virus disease 2019

ZHU Chuanxin, ZHOU Yuping, HE Yan, ZHOU Qin

2021, 36(2):  190-193.  DOI: 10.3969/j.issn.1673-8640.2021.02.014

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Objective To investigate the diagnostic roles of neutrophil/lymphocyte ratio（NLR）,C-reactive protein（CRP）,procalcitonin（PCT）,D-dimer（DD） and amino terminal B-type natriuretic peptide（NT-proBNP）in severe corona virus disease 2019（COVID-19）. Methods A total of 105 COVID-19 patients were enrolled. According to COVID-19 diagnostic criteria,these patients were classified into general group（83 cases）and severe group（22 cases）. Fifty healthy subjects were enrolled as control group. The age,sex,white blood cell（WBC）count,the absolute value of neutrophil（NEUT#）,the absolute value of lymphocyte（LYMPH#）,NLR,CRP,PCT,DD and NT-proBNP of the 3 groups were compared. Results The patients in general and severe groups were older than healthy subjectsand were predominantly male. The patients in severe group were significantly older than those in general group（P<0.05）. Compared with general group,WBC count,NEUT#,NLR,CRP,PCT,DD and NT-proBNP were higher in severe group,and LYMPH# was lower（P<0.05）. General group had higher levels of NLR,CRP,DD and NT-proBNP than healthy control group,while WBC count and LYMPH# were on the contrary（P<0.05）. NT-proBNP had the highest diagnostic value for severe COVID-19,and the combined determination had better performance than single determination. The sensitivity,specificity,positive predictive value and negative predictive value of NT-proBNP were 95.45%,93.98%,80.77% and 98.73%,respectively. Conclusion NT-proBNP is of high diagnostic value for severe COVID-19. The combined determination of NT-proBNP,NLR,CRP,PCT and DD has higher value in the diagnosis of severe COVID-19.

An drug resistance analysis on the mycobacterium tuberculosis complex of pulmonary tuberulosis in Yunfu city

LIN Xiaoling, LIANG Lifeng, WU Yingtao, ZHAO Jieming, CAI Qiuyue

2021, 36(2):  194-197.  DOI: 10.3969/j.issn.1673-8640.2021.02.015

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Objective To analyze the characteristics of drug resistance of Mycobacterium tuberculosis complex（MTBC）in patients with pulmonary tuberculosis in Yunfu city so as to guide individualized treatment. Methods A total of 1 517 sputum culture-positive strains were selected from newly visit tuberculosis patients in Yunfu urban area and 4 counties. MTBC was identified by P-nitrobenzoic acid（PNB）culture medium. Meanwhile,anti-tuberculosis drug sensitivity test of 9 drugs（4 first-line drugs and 5 second-line drugs）were conducted by proportional method. Results Of the 1 571 strains, 1 514（96.4%）were identified as MTBC,and 57（3.6%）were identified as Nontuberculous mycobacterium（NTM）. There was 237 strains（15.7%）of drug-resistance MTBC. The drug-resistance pattern was dominated by monoresistance（9.6%）,followed by polyresistance（3.8%）,multidrug resistance（2.2%）,and extensive resistance（0.07%）. The drug resistance rates of the 4 first-line drugs were streptomycin（11.2%）,isoniazid（7.5%）,rifampicin（3.7%）and ethambutol（3.4%）. The drug resistance rates of the 5 second-line drugs were ofloxacin（1.8%）,aminosalicylic acid（1.5%）,capreomycin（0.5%）,protionamide（0.5%）,and kanamycin（0.3%）. In the 4 first-line drugs,ethambutol had the highest drug resistance（67.6%）to multidrug resistance tuberculosis,followed by streptomycin（52.9%）. In the 5 second-line drugs,ofloxacin had the highest drug resistance（23.5%）to multidrug resistance tuberculosis,and the drug resistance rate of Kanamycin was zero. The drug resistance rate of the first-line combination of isoniazid+rifampicin+streptomycin+ethambutol,isoniazid+rifampicin+ethambutol,and isoniazid+rifampicin were 41.2%,26.4% and 20.6%. The most common combination of multidrug resistance tuberculosis second-line drugs was isoniazid+rifampicin+streptomycin+ethambutol+ofloxacin,and its drug resistance rate was 14.7%. The extensively drug-resistant strain was found in one patient, and it was resistant to all tested drugs. Conclusion The drug resistance of tuberculosis was well controlled in Yunfu city. However, it is necessary to plan personalized treatment based on drug sensitivity test results in vitro,standardize the use of second-line anti-tuberculosis drugs,and prevent drug resistance,especially multidrug resistance and extensively drug resistance.

Survey on the reference intervals of venous blood cells among native Tibetan adults in Aba county

ZOU Ning, CHEN Guangxi, MA Hua, Qiurang, Zhaxinamo

2021, 36(2):  198-201.  DOI: 10.3969/j.issn.1673-8640.2021.02.016

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Objective To establish the reference intervals of venous blood cells among native Tibetan adults in Aba county,Aba Tibetan and Qiang Autonomous Prefecture,Sichuan province. Methods A total of 1 762 healthy native Tibetans in 19 towns in Aba county were enrolled. There were 889 males and 873 females aged 20-79 years old. White blood cell（WBC）count,red blood cell（RBC）count,hemoglobin（Hb）,mean corpuscular hemoglobin（MCH）,mean corpuscular hemoglobin concentration（MCHC）,mean corpuscular volume（MCV）,hematocrit（HCT）and platelet（PLT）count were determined by XN-1000 fully automatic hematology analyzer. The results were compared by gender,and the established reference intervals were compared with the health industry standard WS/T405—2012 Blood Cell Analysis Reference Interval and reference intervals in other regions. Results There was no obvious difference in WBC count and MCV between males and females（P>0.05）. The combined reference intervals were calculated. RBC count,Hb,HCT,MCH and MCHC in males were higher than those in females,while PLT count in males was lower than that in females（P<0.01）. The reference intervals were as follows：WBC count（3.5-9.4）×10⁹/L;RBC count（4.43-6.67）×10¹²/L for males and（4.12-5.88）×10¹²/L for females;Hb 137-204 g/L for males and 125-176 g/L for females;MCH 27-34 pg for males and 25-33 pg for females;MCHC 319-362 g/L for males and 317-355 g/L for females;MCV 81-98 fL;HCT 41%-60% for males and 37%-54% for females;PLT count（153-377）×10⁹/L for males and（150-412）×10⁹/L for females. WBC count,MCV,MCH and MCHC detected had little difference from industry standards,but RBC count,Hb,HCT and PLT count were higher than the industry standard. The reference intervals of this region should be established. Conclusion The reference intervals for venous blood cells among Tibetan adults in Aba county,Aba Tibetan and Qiang Autonomous Prefecture,Sichuan province have been initially established,which can offer accurate evidence for clinical diagnosis, treatment, prognosis and screening of related diseases in this region.

Application of MALDI-TOF MS in methicillin-resistant Staphylococcus aureus rapid identification

GUO Kunshan, WANG Shanmei, MA Bing, XU Junhong, ZHANG Jiangfeng, JING Nan, YAN Wenjuan, MA Qiong, LI Yi

2021, 36(2):  209-212.  DOI: 10.3969/j.issn.1673-8640.2021.02.019

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Objective To discuss the application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry（MALDI-TOF MS） in rapid screening of methicillin-resistant Staphylococcus aureus（MRSA）. Methods Thirty strains of MRSA and 29 strains of methicillin-sensitive Staphylococcus aureus（MSSA）were collected and identified by MALDI-TOF MS. The accuracy of the results was compared. The protein peaks of the bacteria were homologically analyzed by MALDI Biotyper software. The protein peak maps were analyzed statistically by ClinProTools 3.0 software,and the protein peaks with statistical difference were chosen to establish the screening model. Eighty clinical isolates of Staphylococcus aureus（40 MRSA and 40 MSSA）were selected to validate the model. Results The accuracy rate of MALDI-TOF MS in the identification of Staphylococcus aureus was above 99.9%. The protein peaks with charge-to-charge ratios of 6 812,17 668,7 594,and 11 990 m/z were the most important characteristic peaks used to distinguish MRSA from MSSA. The receiver operating characteristic （ROC）curve showed that the area under curve（AUC）of the above four peaks was all 1. Gel scan showed that the protein strength of MSSA group in its four peaks was higher than that of MRSA group. The combination of these four peaks was used to screen 80 tested strains,which showed sensitivity of 95.0%,specificity of 82.5%,positive prediction of 84.4% and negative prediction of 94.3%. Conclusion MALDI-TOF is fast,accurate and low-cost in the identification of Staphylococcus aureus. The differential peaks of MRSA and MSSA can be used to identify the two strains quickly,which provides timely and reliable basis for clinical prevention and treatment of MRSA.

Evaluation of surrogate matrices for serum 25-hydroxyvitamin D calibrators by LC-MS/MS

LI Bingling, CHENG Yating, ZHAO Beibei, SHE Xuhui, DONG Heng, YU Mujun

2021, 36(2):  213-218.  DOI: 10.3969/j.issn.1673-8640.2021.02.020

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Objective To explore the selection and evaluation of a suitable surrogate matrix for preparing 25-hydroxyvitamin D [25（OH）D] calibrators by liquid chromatography tandem-mass spectrometry（LC-MS/MS）. Methods 4% bovine serum albumin（BSA） buffer and 30% ethanol-water were used to prepare 25（OH）D calibrators. They were thoroughly assessed by comparing the peak areas of internal standards in different matrices,testing their matrix effects and accuracy validation. Results There was no significant difference in response between 4% BSA matrix and serum（P>0.05）,while there was statistical difference between 30% ethanol water and serum（P<0.05）. The validation of matrix effect showed that 4% BSA-based calibrator was acceptable,while 30% ethanol-water-based calibrator was not. The external quality assessment（EQA） samples quantified by 4% BSA-based calibrator were well consistent with the target values,but the EQA samples quantified by 30% ethanol-water-based calibrator did not meet accuracy requirements. The bias between the two samples was over 25%. Conclusion 4% BSA can be used as substitute calibrator matrix for the measurement of serum 25（OH）D by LC-MS/MS.

Analysis on the external quality assessment results for 25-hydroxyvitamin D of Shanghai from 2015 to 2019

FANG Huiling, GE Danhong, ZHU Yuqing, ZHU Lingfeng, ZHAO Xiaojun, CHEN Ziqi, WANG Zimeng

2021, 36(2):  219-224.  DOI: 10.3969/j.issn.1673-8640.2021.02.021

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Objective To analyze the external quality assessment（EQA） results for 25-hydroxyvitamin D[25（OH）D] in Shanghai from 2015 to 2019,and to provide experimental data for the improvement of 25（OH）D testing. Methods Ten rounds of EQA results for 25（OH）D in Shanghai Center for Clinical Laboratory（SCCL） from 2015 to 2019 were collected. The means（x）, standard deviation（s） and coefficient of variation（CV） were calculated after eliminating outliers. Results The number of laboratories participating in EQA increased from 41 in 2015 to 83 in 2019. The percent of pass of the 10 EQA in the 5 years were 65.85%,74.70%,82.00%,83.67%,90.91%,87.50%,86.67%,91.89%,92.68% and 92.77%,respectively. The main detection methods were electrochemical luminescence and chemiluminescence,and enzyme-linked immunosorbent assay and liquid chromatography tandem-mass spectrometry were used in a few laboratories. A total of 50 batches were tested in 5 years,among which 33 batches showed a CV less than 20%,6 batches were between 20% and 30%,and the rest were more than 30%. Conclusion There is a large variation of 25（OH）D testing results among different laboratories in Shanghai. It is needed to strengthen the laboratory quality management,to impel the standardization of 25（OH）D testing and to promote the mutual recognition of laboratory test results.