Advantages and challenges of metagenomic next-generation sequencing (mNGS) in the detection of 2019 novel coronavirus / 中华检验医学杂志

As one of the two methods for 2019 novel coronavirus (2019-nCoV), gene sequencing is different from quantitative real-time PCR (RT-PCR) in detection principles. Therefore, gene sequencing has its own pros and cons in clinical application. Currently, metagenomic next-generation sequencing (mNGS) is the most commonly used technology in clinical application. Due to its broad coverage of all types of pathogens, mNGS demonstrates incomparable advantage in rapid identification of novel pathogens such as 2019-nCoV. In addition, it can simultaneously identify other pathogens except 2019-nCoV and mixed infections. On the other hand, however, due to the complexity of mNGS and long detection time, it is unlikely to achieve the purpose of wide-range and rapid diagnosis of 2019 n-CoV. Therefore, mNGS can complement RT-PCR to achieve best clinical application.

Applications and challenges of metagenomic next-generation sequencing in the detection of 2019 novel coronavirus / 中华检验医学杂志

As one of the two methods for 2019 novel coronavirus (2019-nCoV), gene sequencing is different from quantitative real-time PCR (RT-PCR) in detection principles. Therefore, gene sequencing has its own pros and cons in clinical application. Currently, metagenomic next-generation sequencing (mNGS) is the most commonly used technology in clinical application.Due to its broad coverage of all types of pathogens, mNGS demonstrates incomparable advantage in rapid identification of novel pathogens such as 2019-nCoV. In addition, it can simultaneously identify other pathogens except 2019-nCoV and mixed infections. On the other hand, however, due to the complexity of mNGS and long detection time, it is unlikely to achieve the purpose of wide-range and rapid diagnosis of 2019 n-CoV. Therefore, mNGS can complement RT-PCR to achieve best clinical application.

Understanding the influence factors in viral nucleic acid test of 2019 novel coronavirus / 中华检验医学杂志

At present, the prevention and control of new coronavirus has entered a critical period. However, the use of quantitative real-time PCR (qRT-PCR)assays for the detection of viral nucleic acid, as a crucial diagnostic approach, has been doubted in clinical practice. Herein, we have reviewed the current status of epidemic prevention and control, latest development of detection technologies, disease characteristics, clinical sampling and transport. It has also discussed the factors that may affect the performance of viral nucleic acid detection, and suggested some effective methods to improve the detection performance of the assays.

Clinical application of next generation sequencing in molecular diagnosis of intellectual disability/global developmental delay / 中华检验医学杂志

Intellectual disability (ID) is a group of neurodevelopmental disorders with high heterogeneous in both genotypes and phenotypes and its definitive diagnosis is increasingly dependent ongenome-wide molecular diagnostics.Based on next generation sequencing(NGS), panel sequencing, whole exome sequencing (WES) and even whole genome sequencing are well applied to the molecular diagnosis of ID. Based on these, we recommend WES, especially trio-WES as the preferred detection method. NGS data analysis and reanalysis for ID have clinical significance for diagnosis, and can detect small scale variation and copy number variation in the genome reliably. Therefore, it has the potential to become the next recommended molecular diagnostic toolfor ID.

Identifying of the ERCC8 gene mutations in a pedigree with Cockayne syndrome / 中华检验医学杂志

Objective To identify the pathogenesis gene mutation of a pedigree with Cockayne syndrome.Methods The peripheral blood samples of the patient and his family members were collected and the genomic DNA was then extracted.Whole exome sequencing(WES)was performed for proband′s DNA.The disease-causing mutations were identified by bioinformatics analysis and pedigree analysis. Meanwhile,the mutations were confirmed by Sanger sequencing.Results Two novel mutations in ERCC8 gene,including c.400-2A >G and c.394_398delATGTA(p.L132fs)were identified in proband.The splicing mutation originated from his father and changed the splice acceptor site AG to GG, thus possibly caused alternative splicing.The c.394_398delATGTA(p.L132fs)frameshifting mutation was inherited from his mother.The proband′s sister also carried the same compound heterozygous mutation and had the same phenotype as proband.Conclusion The pathogenesis ERCC8 gene mutation of this pedigree with Cockayne syndrome was identified by using whole exome sequencing.

Molecular diagnostics of pediatric neurodevelopmental disorders in children / 中华检验医学杂志

Neurodevelopmental disorders(NDD)is a spectrum of disorders characterized with impaired development of the neuropsychological system and /or functional insufficiency in children.The etiology of NDD is still secure though genetic defects are known to be closely associated with development and prognosis of NDD.Recently, the diagnostic yield of NDD is significantly enhanced along with the application of high-throughput genomic analysis including chromosomal microarray analysis(CMA), target sequencing,and whole exome sequencing(WES)in clinical practice.In view of the high genetic heterogeneity and significant expression variability of NDD, it′s preferred to perform an integrated genetic analysis with multiple molecular diagnostic platforms in a standard workflow for patients with NDD.What′s more,an expert consensus and/or technological guideline for molecular diagnostics of NDD generated from large-scale multi-center studies on cohorts of Chinese patients with NDD is crucial for improvement of healthcare for patients with NDD in China.

The development and prospects of high-resolution melting analysis / 中华检验医学杂志

High-resolution melting analysis ( HRMA/HRM ), a simple, rapid, flexible, inexpensive closed tube approach with high sensitivity and specificity has been one of the most widely used molecular diagnostic techniques in clinicalas well as in research settings .Recently, rapid development ofinstruments , DNA dyes and analysis software significantly enhance the sensitivity , specificity and accuracy of HRM,providing a fast, efficient and economic molecular diagnostic platform for molecular diagnosis of inherited disease , molecular profiling and target therapy of cancer , identification of pathogen , as well as individualized medicine.

Influence of parental origins to the interpretation of chromosomal microarray based clinical pathogenicity analysis / 中华检验医学杂志

Objective To analyze the influence of validating the parental origin to the interpretation of clinical pathogenicity of total 54 copy number variations(CNV)with different clinical significance in 46 patients undergo chromosomal microarray analysis(CMA).Methods A retrospective study.This study enrolled 46 patients conducted in Department of Pediatric Endocrinology and Genetics of Shanghai Xinhua Hospital during the period of August 2014 to December 2015,involving 54 different CNVs detected by CMA.The parental origin of CNVs was examined by CMA or quantitative real-time polymerase chain reaction.Results Totally 54 different CNVs were found in 46 patients by CMA.Seventeen out of the 54 CNVs were pathogenic variations.After validating the parental origin,14 CNVs were proved de novo mutation,while 3 CNVs have maternal origin including 1q21.1 deletion syndrome,Xq27.3q28 and Xq22.1q22.3 duplications which inherited from maternal X chromosome.CNVs of 1q21.1 deletion syndrome often inherited from parents,and no phenotype appears on mother which may be due to the deactivation mechanism of duplications on mother′s X chromosome.Therefore,these 17 pathogenic variations were still considered to be clinical pathogenic significance after validating the parental origin.Ten out of 54 CNVs were variants of uncertain significance-likely pathogenic.After parental original validation,3 CNVs were proved de novo mutation considering likely pathogenic significance,while 7 CNVs have parental origin still judged to be unknown clinical pathogenicity.Twenty-seven out of 54 CNVs were variants of uncertain significance.After validating the parental origin,only 1 CNV was proved de novo mutation considering likely pathogenic significance,while all the others had parental origin considered to be variations likely benign.Conclusion CNVs reported as likely pathogenic should be validated the parental origin in order to further study their clinical pathogenicity,while variants of uncertain significance can preliminary clear its nature by validating parental origin.

The application of chromosomal microarray analysis in genomic diseases / 中华检验医学杂志

Copy number variations in the human genome,one of the causes of complex diseases and genetic diseases,can lead to genomic disorders.As these diseases are difficult to diagnose,it is significantly meaningful to conduct genetic researches and molecular diagnosis.Chromosomal microarray can be used to detect copy number variations on a genome-wide scale.With the advantage of high throughput and resolution,chromosomal microarray is perceived as an important means of identifying copy number variations in genomic disorders.As technology advancements of chromosomal microarray and accumulations of clinical experiences,chromosomal microarray has played a significant role in etiological diagnosis of multiple malformations,mental retardation and autism.

Investigation and analysis of PBL lessons in clinical transfusion science of medical laboratory specialty / 中国输血杂志

Objective To survey and analyze the PBL lessons in the course of Clinical Transfusion Medicine.Evaluate the effect and problems in PBL lessons and explore more effective models.Methods A questionnaire was designed according to the purpose of the survey,4 teachers and 89 students of medical laboratory science in Shanghai Jiaotong University School of Medicine were investigated.Results Students gave high evaluate of both the case and the teachers.Main suggestion was to reduce the class time.When the students were told that the evaluation results will be added to their total score and compared to the teachers' evaluation,they will be more objective.The results were statistically significant.Conclusion PBL cases for Clinical Transfusion Medicine should not be too long.Paying attention to the meaning and feedback of evaluation can greatly improve the enthusiasm and objectivity of the students.

Clinical study of thromboelastography for assessment of coagulation disorders in children with sepsis / 中华急诊医学杂志

Objective To study the clinical significance of thromboelastography (TEG) for determining the presence of coagulation disorders in septic children.Methods A total of 100 patients suffering from sepsis or severe sepsis in pediatric intensive care unit (PICU) of Shanghai Children's Medical Center from February 2014 to January 2015 were recruited.TEG tests and conventional coagulation laboratory tests (CCTs) including platelet count,fibrinogen,prothrombin time (PT),activated partial thromboplastin time,D-dimers,and international normalized ratio (INR) were carried out in all patients at the primary diagnosis of sepsis.Another 25 healthy children taking physical examination were enrolled as control group.Rank Sum Test was used to detect the differences in coagulation markers and TEG between the groups and there was statistical significance when P ＜ 0.05.Receiver operating characteristic (ROC) curves were used to evaluate the roles of TEG and CCTs tests in this study.Results Of them,there were 56 patients with sepsis and 44 with severe sepsis.The male to female ratio was 63∶ 37,the median age was 11.5 (3.3-48) months,and 71％ patients suffered from underlying disease.According to TEG,72 patients had coagulation disorders,including 28 with hypercoagulation and 44 with hypocoagulation.CCTs tests showed 50 patients had coagulation disorders,including 29 with non-overt DIC and 21 with overt DIC.The rate of hypercoagulability was significantly higher in non-DIC group than in non-overt DIC group (46％vs.17.2％,P =0.016).The rate of hypocoagulability was significantly higher in overt DIC group than in non-overt DIC group (100％ vs.44.8％,P ＜ 0.01).Patients with hypercoagulation disorders had significantly shorter R (coagulation reaction time) and K (coagulation formation time) and greater α (angle α),MA (maximal amplitude) and CI (comprehensive coagulation index) compared with control group (P ＜ 0.01).According to CCTs results,patients with hypercoagulation had significantly prolonged PT compared with control group (P =0.002).Compared with sepsis group,severe sepsis group had significantly prolonged R and K and lower α,MA and CI (P ＜ 0.01).ROC analysis demonstrated that area under the curve (AUC) of TEG and CCTs variables for diagnosis of severe sepsis were significantly greater than 0.5.Both variables of α (P =0.000 2) and K (P =0.004 1) had significantly greater AUCs compared with Fib.Conclusions There were 72％ septic patients with coagulation disorders.The hypercoagulability occurred earlier in patients with sepsis and the hypocoagulability occurred later in patients with severe sepsis.The TEG may provide important information for clinicians to deal with coagulation disorders in septic children.

Application of digital PCR for non-invasive prenatal diagnosis of inherited diseases / 中华检验医学杂志

Inherited diseases are characterized with a great variety of clinical entities, complex underlying etiologies, and absence of effective treatment, emerging as one of the significant threats to human′s, esp., the health and wellbeing women and children.It′s long been recognized as a powerful and cost-effective strategy to implement prenatal diagnosis for inherited diseases with an array of advanced molecular diagnostics to reduce the nationwide rate of birth defects.Recently, non-invasive prenatal diagnosis for inherited diseases is increasingly applied in research as well as in clinical practice.Digital PCR is a novel technology characterized with superb sensitivity, high accuracy, and absolute quantitation of DNA, and has demonstrated excellent performance in non-invasive prenatal diagnosis of several hereditary disorders, including spinal muscular atrophy, sickle cell anemia, and hemophilia.It′s believed that digital PCR has more to offer in improving non-invasive prenatal diagnosis of inherited diseases in future.

Application of next generation sequencing technology in molecular diagnosis of rare diseases / 中华检验医学杂志

Due to a wide variety of classification and phenotypes,rare diseases are difficult to be diagnosed in the clinical practice Studies have shown that most rare diseases belong to inherited diseases,so it is particularly important to carry out the genetics research and molecular diagnosis.Next generation sequencing (NGS) technology has the advantages of a high throughput,high sensitivity etc,which is the main research means to identify the pathogenic genes of rare diseases at present.With the development of NGS and the bioinformatics technology,in recent years the whole exome sequencing and targeted panel sequencing have been gradually applied to clinical molecular diagnosis,which is important to increase the accuracy of diagnosis and to improve the therapeutic effect of rare diseases.

Comparison of three methods for the genetic diagnosis of spinal muscular atrophy / 中华检验医学杂志

Objective To evaluate the value of PCR-restriction fragment length polymorphism (PCR-RFLP),real-time PCR and multiplex ligation-dependent probe amplification (MLPA) in the genetic diagnosis of spinal muscular atrophy (SMA) and make laboratory support accessible to clinicians for the molecular diagnosis of SMA.Methods Methodological evaluation.Forty-one suspected SMA cases and 359 control individuals received in Shanghai Children's Medical Centre from March 2013 to June 2014 were detected for the deletion of exon 7 and 8 in the survival motor neuron gene 1 (SMN 1) by PCR-RFLP,realtime PCR and MLPA,respectively.Then the results of the three methods were compared and the benefits and limitations of the three methods were evaluated.Results The result of real-time PCR was in complete agreement with that of MLPA,showing that 29 suspected cases harbored homozygous deletions of SMN1 and 1 case possessed heterozygous deletion.Among the homozygous deletions,27 patients demonstrated absence of exon 7 and 8,and 2 cases demonstrated only the absence of exon 7.Meanwhile,both PCR-RFLP and MLPA analysis showed the same results that only 5 out of 395 control cases carried heterozygous deletion.As for cases without heterozygous deletions,PCR-RFLP demonstrated the same result with real-time PCR and MLPA but it missed all the heterozygous ones.Conclusions PCR-RFLP,the conventional SMA gene diagnosis method,was only capable of detecting homozygous deletion of exon 7 and/or 8 of SMN1,but was not as sensitive as to find out the carriers with heterozygous deletions.MLPA could detect the deletion and quantify the copy numbers of exon 7 and 8 of SMN1,efficiently,while the price was relatively high,which brings challenges for its application in the carrier screening of SMA.Compared with these two methods,realtime PCR with high throughput and low input was a rapid,acourate and economic method for the genetic diagnosis of SMA and carrier screening in large populations.

The functional study of antithrombin L99 mutation / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the molecular mechanisms of inherited antithrombin (AT) deficiency caused by AT L99 mutation.</p><p><b>METHODS</b>Wild type (WT), L99V, L99A, L99I and L99S AT were purified from drosophila expression system. The binding capacity of AT and the low molecular weight heparin sodium was analyzed by the heparin binding assay. Surface plasmon resonance (SPR) was used to detect the binding ability of AT to thrombin (FIIa) or AT to coagulation factor Xa (FXa). The activity of AT(AT∶A)was detected by chromogenic assay.</p><p><b>RESULTS</b>The purified WT and mutant AT were at the same size. No additional band was observed by coomassie blue staining and western blot assay. Compared to the WT AT, the binding abilities of the low molecular weight heparin sodium to the AT L99V, L99A, L99I and L99S were (44.8±3.6)%, (118.9±14.0)%, (15.2±8.8)%, and(23.0±8.2)%, respectively. The binding abilities of FIIa to AT L99V, L99A, L99I and L99S were 13%, 57%, 3%, and 29%, while the binding of FXa to AT L99V, L99A, L99I and L99S were 7%, 51%, 1%, and 25%. The AT∶A of WT, L99V, L99A, L99I and L99S AT were 146.5%, 21.4%, 120.9%, 10.8%, and 39.0%, respectively.</p><p><b>CONCLUSION</b>The binding abilities of AT to heparin, FIIa and FXa were damaged by the L99 mutation, which resulted in decreased AT∶A and inherited AT deficiency.</p>

Laboratory screening and diagnosis of inherited metabolic diseases in the newborn / 中华检验医学杂志

Birth defect is increasingly an issue of public health and social concern.Newborn screening is the principal content of 3-tiered system of prevention and control for birth defects in China,which plays an important role in promotion of children's health and welfare.Widespread application of mass spectrometry,esp.,tandem mass spectrometry in newborn screening of inherited metabolic diseases has greatly contributed to the increased detection capability and efficiency.Low and medium throughput molecular diagnostic techniques including PCR,Sanger sequencing,high resolution melting analysis,and multipleligation dependent probe amplification are widely applied in diagnosis and discrimination of inherited metabolic diseases.Application of next generation sequencing in newborn screening is emerging,and will undoubtedly revolutionize the arena of newborn screening in future.However,vigorous validation and performance evaluation are warranted before it's applied in newborn screening for inherited metabolic diseases.

A Missense Mutation of IDS Gene in A Chinese Child with Mucopolysaccharidosis Type Ⅱ / 现代检验医学杂志

Objective To study the clinical characteristics and iduronate-2-sulfatase (IDS)gene mutation of one child patient with mucopolysaccharidosis type Ⅱ (MPS Ⅱ).Methods All the 9 exons of IDS gene were amplified by polymerase chain reaction (PCR)technlogy.The PCR products were screened by direct gene Sanger sequencing.Results A missense muta-tion (c.445T>C)on exon 4 was found after the analysis of the gene sequencing results of PCR products in this patient’s IDS gene.Thi smutation leaded to the 149th codon TCT encoded serine into a CCT encoding proline (p.Ser149Pro).Mean-while,the IDS gene in the parents were widetpye,so this was a de novo mutation.Conclusion The de novo mutation of IDS gene is the cause of our patient with?mucopolysaccharidosis,one novel mutation (p.Ser149Pro)was identified.

Investigation and analysis on reference value range of peripheral blood routine in preschool children from Shanghai / 国际检验医学杂志

Objective To investigate and understand the reference value range of hematological parameters for peripheral blood routine in preschool children from Shanghai.Methods The Sysmex XS-800i automated hematology analyzer and the original rea-gents were used to measure the hematological parameters in peripheral blood samples collected from 7 692 healthy preschool chil-dren aged 2-6 years in Shanghai,including white blood cells(WBC),red blood cells(RBC),platelet(PLT)count,hemoglobin(Hb), hematocrit(HCT),mean corpuscular volume(MCV),mean corpuscular hemoglobin(MCH)and mean corpuscular hemoglobin con-centration(MCHC);the various parameters in different age groups were collected and statistically processed to establish the refer-ence intervals for each parameter.Results In the blood routine among preschool children in Shanghai,there were statistically signif-icant differences in all parameters except WBC and PLT count between sexes among preschool children in Shanghai(P <0.05).The reference intervals of hematological parameters obtained in this investigation were obviously differed from those offered by the man-ufacturer.However,compared with those from the related reports,the difference existed in partial parameters,the upper limit of WBC count was highest compared with the results from some areas,while the reference value ranges of MCV,MCH and MCHC were higher than those of other area study results.Conclusion The independent blood routine medical reference value range for preschool children in Shanghai should be established and the influence of the factors of gender,instrument and reagents also should be taken into consideration in establishing the reference value range.

Pedigree analysis of a child with neonatal diabetes / 临床儿科杂志

10.3969/j.issn.1000-3606.2013.06.019

Chromosomal microarray analysis in prenatal diagnosis / 中华检验医学杂志

Prenatal diagnosis is an effective approach for preventing birth defects and improving population health.Chromosomal karyotyping,sonography,serum screening,fluorescence in situ hybridization,and PCR-based techniques are examples of current prenatal diagnostic technologies.In recent years,the clinical utility of chromosomal microarray analysis (CMA) have been well demonstrated in postnatal genetic diagnosis and it has been recommended as the first tier test for global developmental delay,mental retardation,congenital multiple anomaly,and autism spectrum disorders.CMA is now also being applied to prenatal testing.However,there are still many unresolved issues regarding the proper use of CMA in prenatal testing.The issues include but not limit to the clinical indications for prenatal CMA,interpretation for copy number variations of unknown significance,selection of array platforms,and genetic counseling.These issues should be addressed in order to properly use CMA in prenatal diagnosis.We believe close collaboration from professionals of different disciplines involved in patient care is necessary to help establish the clinical guideline and best practice recommendation for application of CMA in prenatal diagnosis.