Thinking of the current status and application of machine learning in the field of laboratory medicine / 中华检验医学杂志

In recent years, machine learning has become a hot spot in various research fields. Using machine learning can realize the transformation from data driven to knowledge discovery, which is an important development direction of laboratory intelligence in the future. The application of machine learning in laboratory medicine has shown great potential, but t it also has many challenges and difficulties. The direction of our joint efforts is to promote the clinical transformation of machine learning technology, realize the practicality and industrialization in medical laboratories, and achieve the goal of assisting clinical decision-making as soon as possible.

Application of deep learning in antinuclear antibodies classification: progress and challenges / 中华检验医学杂志

Antinuclear antibodies (ANA) testing is essential for the diagnosis, classification, and disease activity monitoring of systemic autoimmune rheumatic diseases. In recent years, with the enhancement of computing power and the innovation of algorithms, the newly hip branch, deep learning (DL), practically delivered all of the most stunning achievements and breakthroughs in artificial intelligence (AI) so far. The application of DL to visual tasks, known as computer vision, has revealed significant power within the medical image recognition. Indirect immunofluorescence on HEp-2 cells is the reference method for ANA testing, the results is interpreted manually by specialized physicians. ANA fluorescent pattern classification is based on image recognition, which has a broad prospect of combining with DL to realize automatic interpretation system. This paper reviews the recent research progress and challenges of DL in the field of ANA detection in order to provide references for the standardization of ANA testing in the future.

Current state and future prospect of autoimmunity laboratory diagnosis / 中华检验医学杂志

Laboratory testing is of great value in the management of autoimmune disease. The results can help confirm a diagnosis, estimate disease severity, aid in assessing treatment effect. But the current autoimmunity laboratory system, including testing standards, quality control and supervision, does not match the national conditions well. As a result, the test reports are not mutual-recognized among laboratories. In the current background of precision medicine, with the advances of technology and the application of deep learning and artificial intelligence in the clinical laboratory field, the autoimmune laboratory has ushered in a new development trend of integration, automation and intelligence.

Current state and future prospect of autoimmunity laboratory diagnosis / 中华检验医学杂志

Laboratory testing is of great value in the management of autoimmune disease. The results can help confirm a diagnosis, estimate disease severity, aid in assessing treatment effect. But the current autoimmunity laboratory system, including testing standards, quality control and supervision, does not match the national conditions well. As a result, the test reports are not mutual-recognized among laboratories. In the current background of precision medicine, with the advances of technology and the application of deep learning and artificial intelligence in the clinical laboratory field, the autoimmune laboratory has ushered in a new development trend of integration, automation and intelligence.

The status quo and future prospects of the next generation sequencing technologies in clinical diagnostics / 中华检验医学杂志

During the past decade, tremendous progress has been made in terms of speed, read length, and throughput, along with a sharp reduction in per-base cost.Together, these advances democratized next generation sequence (NGS) and paved the way for the development of a large number of novel NGS applications in clinical diagnostics, especially in the field of non-invasive prenatal detection, rare genetic disease and cancer companion diagnostics.As technology advances, long-read single molecule sequencing began to emerge.Single cell, long-reads, transcriptome, and low cost will be the NGS direction.Due to the special nature of clinical testing, the current NGS clinical application system,including genetic counseling, testing standards, quality control, supervision, database construction etc, does not match the national conditions well and still faces a few challenges, needs to be constantly improved through the routine clinical practice in the future.

Analysis of clinical phenotype and genotype of two hereditary coagulation factor Ⅻ deficiency / 中华检验医学杂志

Objective Two Chinese pedigrees with congenital factor Ⅻ(FⅫ)deficiency were enrolled in the present study,and studies on the clinical manifestations,family survey,biochemical examinations and gene diagnosis of these pedigrees were performed.Methods In October 2014-2015 March,two cases of hereditary FⅫ deficiency patients were included in Xinhua hospital.Activated partial thromboplastin time(APTT),FⅫ procoagulant activity(FⅫ:C),FⅫ antigen(FⅫ:Ag)and other parameters of coagulant were detected.The FⅫ deficiency pedigree members,exons 1-14,boundary introns including the splice junctions of the F12 gene were amplified with polymerase chain reaction(PCR).Direct sequencing was exerted to purified PCR product to detect the gene mutation.If the gene mutations were found,polymorphism should be ruled out by directing sequence.One hundred and three healthy persons as normal controls.Results The two probands were manifested prolonged APTT(101 s and 143 s).They showed lower FⅫ activity and FⅫ antigen(2％and 6％,0.4％and 4％,respectively).FⅡ:C,FⅦ:C,FⅧ:C,FⅨ:C,FⅩ:C and Fg are normal in the two probands.LAC is negative.Proband 1 has c.1285C>T(p.Q429 stop)mutation.His parents and son have the heterozygous mutation in the same position.Proband 2 has c.1556T>C(p.L519P)mutation.Her two sons have the heterozygous mutation in the same position.In the promoter regions of F12 gene,there were common 46C/T and 619 G/C polymorphisms in two pedigrees.Conclusion c.1285C>T(p.Q429 stop)and c.1556T>C(p.L519P)are the cause of FⅫ deficiency.

Molecular diagnosis in China：present status and challenges ahead / 中华检验医学杂志

Molecular diagnosis is rapidly developed in recent years , mainly applicated in the diagnosis of hereditary disease , infectious pathogens, tumor susceptibility and molecular typing , companion diagnosis and prognosis assessment , playing more and more important role in many diseases diagnosis and treatment.Molecular diagnosis was developed from the eighties of the last century in our country .Nowadays, the mainly applied technologies in the clinical laboratory include fluorescence in situ hybridization , quantitative PCR, microarray and DNA sequencing. These molecular technologies make up for the insufficiency of routine testing and take up a central role in the development of modern laboratory medicine . With the continuous development in transformation research of molecular technology recent years , there will be more molecular diagnostic techniques applied in clinicaldiagnosis in the future .But it still exists some drawbacks in the performance of molecular diagnosis in our country according to the current situation , such as imbalanced regional development , mismatched policies, non-standardized laboratory construction , deficiency of quality control and supervision , etc., which requires the joint effort of the government , hospital, professional association and clinical laboratory itself to promote the healthy and orderly development of molecular diagnosis.

Comparison of three different flow cytometers in the clinical assessment of lymphocyte subsets / 中华检验医学杂志

Objective To evaluate the consistency and accuracy among 3 brands of flow cytometers (BriCyte E6,BD FACSCanto Ⅱ and Beckman Coulter FC 500) in the detection of lymphocyte subsets.Methods According to the methodology,the BriCyte E6 was compared with 2 flow cytometers commonly used in clinical detection.Seventy-three cases (40 male and 33 female) of anticoagulation peripheral blood specimens were collected in the clinical laborartory department of Xinhua Hospital in July 2015 and the percentage (％) and absolute number (#) of the lymphocyte subsets were detected by 3 different flow cytometers within samples collected 4 h.Results There were good consistency among the 3 flow cytometers (R2 ＞0.95,R2 from 0.969 5 to 0.992 4) in the detection of lymphocyte subsets percentage,so did in the detection of absolute number (R2 ＞ 0.95,R2 from 0.969 1 to 0.993 3).As to the precision evaluation,in the detectionof CD8％,T#,CD4+ T# and CD8+ T#,BriCyte E6 achieved a low CV％ compared with FACSCanto Ⅱ and FC 500 (Friedman statistics are 16.720,11.840,15.760 and 15.430,P =0.000 2,0.027,0.000 4,0.000 4,respectively).In the detection of T％,CD4％,NK％,B％,NK#,B#,there was no significant difference among the 3 flow cytometers (Friedman statistics are 4.242,3.916,0.852,2.595,1.835 and 0.578,P =0.119 9,0.141 2,0.653 2,0.273 3,0.399 6,0.749 0,respectively).Conclusions The 3 flow cytometers have a good consistency in the detection of lymphocyte subsets.BriCyte E6 may be an alternative or complement of existing flow cytometers.

The status quo and prospects of flow cytometry clinical application in China / 中华检验医学杂志

Flow cytometry (FCM) is one of the most advanced cell quantitative analysis technology.Today,flow cytometry has been extensively and intensively used in medicine and other science,ranging from basic research to clinical diagnosis With the rapid development of science and technology of China,the application of flow cytometry in clinical research and diagnosis has also made significant progress.

The correlation between atypical respiratory pathogens infection and serum total IgE levels / 国际检验医学杂志

Objective To investigate the correlation between atypical respiratory pathogens infection and serum total IgE levels . Methods Serum IgM level was detected in 1 913 blood samples of children with atypical respiratory infection by using indirect im‐munofluorescence assay ,including mycoplasma pneumonia(MP) ,legionella pneumophila (LP) ,rickettsia Q(QFR) ,chlamydia pneu‐monia(CPn) ,adenovirus (Adv) ,respiratory syncytial virus (RSV) ,influenza A virus (IAv) ,influenza B virus (IBv) and parainflu‐enza virus (PIV)1/2/3 .The serum total IgE level was detected by immune scatter turbidimetry .Software SPSS 17 .0 was used in data statistical analysis .Results A total of 991 out of 1 913 samples of respiratory inflected children exhibited positive(positive group) ,while 922 exhibited negative(negative group) in indirect immunofluorescence assay .650 out of the 991 positive samples (65 .59% ) contained MP infection and the combination of MP infection and other virus infections .The serum total IgE level in posi‐tive group was significantly higher than that of the negative group ,and the serum total IgE level in samples with MP infection was higher than that in samples with IBv infection ,Adv infection ,and RSV infection .In the samples in which serum total IgE level was higher than the clinical reference range (100 kU/mL) ,the infection rate of MP infection alone was 31 .29% ,which was evidently higher than that in samples of low IgE level(< 100 kU/mL ,21 .30% ) .On the other hand ,the infection rate of RSV alone was 1 .88% and the infection rate of Adv alone was 3 .13% ,which were both evidently lower than those in samples with normal serum total IgE level(both 6 .53% ) .Conclusion MP is the most common pathogen in children with atypical respiratory pathogen infec‐tion ,and can lead to higher serum total IgE levels .

Application evaluation of immunophenotyping in monoclonal gammaglobulinemia / 国际检验医学杂志

Objective To investigate the clinical value of immunophenotyping and quantitative analysis of serum immunoglobulin in the diagnosis and typing of monoclonal gammaglobulinemia.Methods 118 serum samples were collected from the patients with monoclonal gammaglobulinemia and performed the serum protein electrophoresis(SPE),serum immunofixation(IF)electrophoresis, immunoglobulins(Ig)quantitation and serum light chain κ,λdetection.The analysis was conducted aiming at the patients with im-munophenotyping positive and the Ig quantitation negative.Results 56 cases showed immunophenotyping positive and the immuno-globulin quantitation negative,among them,6 cases were free light chain type (κlight chain in 2 cases andλlight chain in 4 cases), 1 case was the non-secretion type;62 cases showed immunophenotyping positive and the Ig quantitation positive.Conclusion The IF technique has an important significance for the diagnosis and typing of monoclonal gammaglobulinemia,its diagnosis can be com-bined with the other laboratory tests of serum Ig and light chain quantitative detection.

Comparison of yeast identification ability between Bruker Microflex MALDI-TOF MS and Vitek 2 Compact automatic microbial analysis system / 中华检验医学杂志

Objective To evaluate and compare yeast identification ability between Bruker Microflex matrix-assisted laser desorption ionization-time of flight mass spectrometry( MALDI-TOF MS) and Vitek 2 Compact automatic microbial analysis system.Methods Retrospective study.Totally 742 strains of yeast isolated from clinical specimens during March 2013 to March 2014 in Xinhua Hospital, Shanghai Jiao Tong University School of Medicine were identified by Bruker Microflex MALDI-TOF MS and Vitek 2 Compact automatic microbial analysis system simultaneously.The strains with discordant results were validated by gene sequencing.Results The coincidence rate of 699 Candida identified by Bruker Microflex MALDI-TOF MS or Vitek 2 Compact system was 100.0%(699/699) and 99.6%(696/699) to the species level, respectively and the coincidence rate of 43 yeast-like fungi strains identified was 90.7%(39/43) and 79.1%(34/43) to the species level, respectively.Penicillium marneffei could not be identified by both two instruments, but protein profile of Penicillium marneffei by MALDI-TOF MS was established.Conclusions The coincidence rate of yeast identified by Bruker Microflex MALDI-TOF MS is higher than that of Vitek 2 Compact system.Using Bruker Microflex MALDI-TOF MS to identify yeast especially Candida and yeast-like fungus is fast, simple, low-cost, accurate, and it can be used in routine work of ordinary yeast identification in clinical microbiology laboratory.

Development and validation of a liquid chromatography-tandem mass spectrometry method for the quantification of creatinine-corrected sarcosine in urine / 中华检验医学杂志

Objective To establish a liquid chromatography-tandem mass spectrometry ( LC-MS/MS) method for the quantification of creatinine-correctedsarcosine in urine for the prostate cancer diagnosis and treatment.Methods It performed the method establishment and evaluation in this study.Random unrine samples were collected from 36 subjects with prostate cancer, 15 subjects with benign prostatic hyperplasia and 76 healthy people receiving medical examination.Urine samples mixed with [ 2 H3 ]-labeled sarcosine were treated by precolumn derivation using dansyl chloride, then analyzed by LC-MS/MSsystem in multiple reaction monitor ( MRM) mode.Sarcosine and creatinine were quantified by the isotope internal standard method and the standard curve was employed with a series of calibration.The limit of detection, precision and recovery were also evaluated in this study.The results of this methodology were compared with those of the enzymatic method.Results Sarcosine could be distinguished against its isomers completely. The linear equation of sarcosine was Y=2.045 6X+0.068 9, R2 =0.994.The limit of detection and limit of quantity were 8 ng/ml and 25 ng/ml respectively.The intraassay and interassay coefficients of variation were both below 6%.The recovery ratio of sarcosine ranged from 96.8%to 105.1%.The results from the ID-LC-MS method correlated with those from enzymatic method (R2 =0.815, P <0.01).Compared to enzymatic method, the average bias of sarcosine was -37.1%.Conclusions It established a LC-MS method for urinary sarcosine quantification with good specificity, sensitivity and repeatability.This method can provide a reliable platform for the diagnosis of prostate cancer.

The detection and clinical application of serologic biomarkers for liver fibrosis, the present and future perspectives / 中华检验医学杂志

Hepatic fibrosis is the final common pathway for liver injury caused by various chronic liver diseases.Noninvasive assessments of liver fibrosis are valuable in clinical use since they are easy to apply and monitor disease progression.Serologic biomarkers for liver fibrosis consist of direct markers which reflect ECM turnover and indirect markers which show liver function of synthesis,metabolism and reservation.A number of assessment models can identify significant fibrosis and cirrhosis,and avoid unnecessary liver biopsy.They can be used for disease staging,prognosis prediction,and treatment surveillance.Variation of analytical performances exists among diverse analyzers and assays.The standardization of these tests will enhance their comparability.Large scale and multicenter clinical verification studies are needed for the clinical utility of these assessment models based on Chinese population of chronic hepatic diseases.

The carbapenem-resistance mechanism and homology analysis of four strains of Klebsiella pneumoniae isolated from different sites of one patient / 中国感染与化疗杂志

Objective To study the drug resistance mechanism and homologous relationship of four clinical strains of carbapen-em-resistant Klebsiella pneumoniae isolated from different sites of the same one patient.Methods Four carbapenem-resistant K .pneumoniae strains were isolated from blood,urine,sputum and pelvic drainage of a patient after cystectomy in clinical mi-crobiology lab of Xinhua Hospital in March 2012.① Modified Hodge test was used to detect the expression of carbapenemase.② PCR amplification assay and DNA sequencing were used to detect resistance-related genes.③ SDS-PAGE analysis was per-formed to analyze outer membrane porin components of the four carbapenem-resistant K .pneumoniae isolates.ERIC-PCR as-say was used to analyze the homology of these carbapenem-resistant isolates.Results Modified Hodge test demonstrated that all the four carbapenem-resistant K.pneumoniae expressed carbapenemase.KPC-2 gene was identified in all the four isolates by PCR test and DNA sequencing.SDS-PAGE analysis indicated an outer membrane porin alteration common to all the four iso-lates which is distinct from that of the strain sensitive to antibiotics.The identical DNA fingerprinting of the four strains was confirmed by ERIC-PCR analysis.Conclusions The antibiotic resistance mechanism of the four carbapenem resistant K.pneu-moniae associated with this case includes the expression of KPC-2 and altered outer membrane permeability induced by abnormal expression of outer membrane porin.These four strains belong to one common clonal group.

Genotyping of K-ras exon 2 codons 12 and 13 mutations in colorectal cancer by pyrosequencing / 中华检验医学杂志

ObjectiveTo investigate the clinical significance of pyrosequencing assay for determining K-ras mutations in exon 2 codons 12 and 13 in clinical colorectal cancer tissues.Methods Genomic DNA,extracted from K-ras mutant cell lines SW480 (homozygous,c.35G ＞ T), DLD-1 (heterozygous,c.38G ＞ A) and wild-type HT-29,was first used as the sequencing template respectively to test the accuracy of pyrosequencing methodology.The SW480 and DLD-1 DNA was separately mixed with wild-type HT-29 DNA in proportions of 2％,3％,5％,10％,20％,30％ and 50％,the sensitivity for mutation detection was measured separately by pyrosequencing assay and directed Sanger DNA sequencing in the serial DNA mixture samples.The pyrosequencing assay results were compared with the corresponding Sanger sequencing and the datas were analysized by Fisher exact test.Pyrosequencing analysis was then performed for screening K-ras exon 2 mutations at codons 12 and 13 on DNA isolated from a panel of 30 colorectal cancer samples derived fromclinicalformalin-fixed andparaffinembedded(FFPE)tissues.ResultsCancer cell lines with known K-ras mutations ( SW480 and DLD-1 ) were readily detectable by pyrosequencing-based analysis.When the proportions of mutant colorectal cancer cell line DNA were 5％ and 10％ content,the mutation rates of K-ras gene detected by conventional Sanger DNA sequencing were 33.3％ (4/12) and 58.3％ (7/12) respectively,whereas the mutation rates detected by pyrosequencingbased assay were 91.7％ (11/12) and 100％(12/12) respectively,there were significant differences between those two sequencing methodology ( P ＜0.05).Furthermore,we found 10 patients with K-ras exon 2 point mutations at codons 12 and 13 by pyrosequencing-based assay from 30 colorectal cancer FFPE tissues,the point mutation rate was 33.3％ (10/30) and all of the mutations determined were heterozygous.The codon 12 was most frequently affected [30％ (9/30)].Mutations with the highest frequency were G ＞ A transitions [ 50％ ( 5/10 ) ],followed by G ＞ T transversions [ 30％ ( 3/10 ) ].Conclusion The pyrosequencing assay provides an accurate and sensitive method for mutation screening of K-ras exon 2 codons 12 and 13 in routine diagnostic specimens,thereby allowing the selection of the cancer treatment in clinical individualized practice.

Extending the clinical application and improving the practical techniques of flow cytometry / 中华检验医学杂志

With the improvement of the multi-parameter and high throughput technology,FCM has expanded its clinical application from cellular phenotying to other areas,such as protein function analysis,fine needle aspiration,cerebrospinal fluid determination,circulating tumor cell detection as well as clinical microbiology.Currently,lack of standards for the sample processing,equipment adjustment and data analysis remains the main obstacle for routine application of FCM.Therefore,it is recommended to establish consensus standard operation procedures as well as shared FCM platform regionally to provide better service of FCM in clinical laboratory.

Analysis of serum chemokines in patients with gastric cancer by cytometric bead array and its clinical significance / 中华检验医学杂志

Objective To explore the levels of serum chemokine in the patients with gastric cancer using cytometric beads array (CBA) and to find out the laboratory evidence for gastric cancer immunotberapy.Methods Forty-five patients with gastric cancer,thirty patients with benign gastric diseases and forty healthy controls were included.The chemokine levels of interleukin-8 (IL-8),regulated upon activation normal T-cell expressed and secreted (RANTES),monokine induced by IFN-γ (MIG),monocyte ehemoattractant protein-1(MCP-1) and interferon-γ induced protein-10 (IP-10) in serum of these three groups were measured using CBA ,then these data were analyzed using BD CBA analysis software.Results The levels of serum chemokines in healthy controls were (176.4±20.7) μg/L for IP-10,(111.3±17.2) μg/L for MCP-1,(503.9±47.2) μg/L for MIG,(472.4±116.7) μg/L for IL-8,respectively.In gastric cancer patients,the levels were (266.6±24.7) μg/L for IP-10,(100.4,70.8-193.5) μg/L for MCP-1,(1614±275.4) μg/L for MIG,(500.0±164.8) μg/L for IL-8.The levels of serum chemokines in patients with benign gastric disease were (207.9±31.7) μg/L for IP-10,(121.2±23.6) μg/L for MCP-1,(514.5±63.0) μg/L for MIG,(480.2±134.8) μg/L for IL-8,respectively.The levels of IP-10 and MIG among these three groups were significantly different (IP-10:F = 3.52,P < 0.05,MIG:F = 9.27,P < 0.01).The levels of IP-10 were elevated in the cancer patients compared with healthy controls (P < 0.05),while the levels of MIG were elevated significantly in the cancer patients compared with healthy controls and benign disease controls(t=3.29,P < 0.01,t=2.84,P<0.01).Conclusions CBA is a novel method with convenience,sensitivity and accuracy for the detection of serum ehemokines.The concentration of IP-10 and MIG in gastric cancer patients are elevated,indicating its implication in cancer pathogenesis and metastasis.Measurement of serum chemokines will lay the groundwork for therapeutic strategy in gastric cancer by inhibiting and anti-chemokine.

Investigation on gene defects in a pedigree with inherited coagulation factor Ⅺ deficiency / 中华检验医学杂志

Objective To investigate the gene defects of a pedigree with inherited coagulation factor Ⅺ (FⅪ) deficiency by analyzing its phenotype and molecular genetic characteristics. Methods A pedigree with inherited FⅪ deficiency was enrolled in this study. The activated partial thromboplastin time (APTF), prothrombin time (PT), FⅪ activity (FⅪ: C) and FⅪ antigen (FⅪ: Ag) were determined for phenotype diagnosis. Fifteen exons and their flanks of F11 gene from the proband's genomic DNA were amplified by polymerase chain reaction (PCR), and the PCR products were directly sequenced to analyze the F11 gene mutation. The PCR products amplified from genomic DNA from the proband, her parents and 100 healthy donors were digested with restriction enzyme BssSI to exclude gene polymorphism and confirm the mutation site. The cleavage site in the signal peptide was predicted by the SignalP software. Results The values of APTT, PT, FⅪ: C and FⅪ: Ag of the proband were 69.5 s, 12.3 s, 2.6% and 2.5%, respectively, indicating that this case was cross-reacting material (CRM) negative. The same values of healthy controls were 35 s, 13 s, 100% and 100%, respectively. As compared with Genbank AY191837 sequence, four variants in F11 exons were found. G3733C heterozygous mutation in exon 2 causod Gly to Arg substitution at-1 amino acid position in signal peptide (G-1R). The G3733C mutation in exon 2 introduced a new BssSI enzyme digestion site. Further analysis of the 100 randomly collected DNA samples from the normal population excluded the possibility of G3733C as a polymorphism. CI6642T heterozygous mutation in exon 8 introduced a premature stop codon at 263 amino acid position (Q263Term). Conclusions G-1R mutation and Q263Term compound heterozygous mutation in F11 gene are the mechanism of FⅪ deficiency for the proband. G-1R mutation is a novel F11 gene mutation causing inherited FⅪ deficiency.

The influences on carbohydrate-deficient transferrln level in healthy individuals after short term alcohol intake / 中华检验医学杂志

opping drinking. They were not influenced by gender, smoking and drinking histories. They could serve as monitoring indexes for recent drinking status on healthy individuals.