Application prospect of nuclear magnetic resonance spectroscopy in clinical laboratory examination / 中华检验医学杂志

Nuclear magnetic resonance spectroscopy (NMRS) is a branch of spectroscopy, which can be used to determine the number, type and relative position of components in the mixture. Due to its high throughput, high sensitivity and high stability, especially its "fingerprint", non-destructive and non-biased detection of metabolites, NMRS has become one of the most commonly used analytical and detection techniques in metabolomics. Based on the research of clinical laboratory application, this review briefly expounds the technical principle of nuclear magnetic resonance spectroscopy, introduces the development and latest research results of nuclear magnetic resonance spectroscopy in biomedical application fields such as blood lipid analysis, tumor detection, prediction of mental and nervous system diseases, infectious diseases, nutrition and health management, and discusses the development prospect of clinical translational medicine.

Research progress of extracellular vesicle metabolomics in tumor / 中华检验医学杂志

Extracellular vesicles (EVs) can carry a variety of bioactive components including nucleic acids, proteins and small molecule metabolites, and their value in tumor diagnosis and treatment has been widely recognized. However, current studies on EV inclusions mainly focus on RNA and protein, and the role of small molecule metabolites that can most directly reflect the cell state in EV remains unclear. EV metabolomics in cancer research has gradually gained traction in recent years. There are still many challenges in EV metabolomics research due to the complexity of pretreatment and low content of metabolite, but its value in regulating tumor progression and serving as tumor markers has gradually emerged, which is expected to provide new targets for tumor diagnosis and treatment.

Application progress of metabolomic analysis based on nuclear magnetic resonance spectroscopy in tumor diagnosis and treatment / 中华检验医学杂志

Nuclear magnetic resonance spectroscopy is one of the main analytical techniques for detecting metabolomics, which has the advantages of simple operation, rapid detection and non-invasive feature. By monitoring the changes of metabolites in the body, it is helpful to deeply understand the mechanism of disease and play a role in the diagnosis and treatment of diseases, but its clinical application has not yet been popularized. In recent years, the application of metabolomics in tumors has increasingly become a research hotspot. Therefore, in order to provide a reference for the research and clinical application of tumor metabolomics, the nuclear magnetic resonance spectroscopy and tumor metabolomics were introduced in this paper, and the application progress of metabolomics analysis based on this technique in early tumor screening, clinical diagnosis and prognosis evaluation were reviewed in this paper.

Establishment principles and research progress on patient based real time quality control / 中华检验医学杂志

Patient based real time quality control (PBRTQC) is a quality control method that uses the test results of clinical specimens from patients to monitor the analysis performance of the test process in real time and continuously. Although the International Federation of Clinical Chemistry and Laboratory Medicine PBRTQC working group had recommended that this method should be popularized in clinical practice in 2020, there is still certain lagging in cognition, research and application of PBRTQC in domestic clinical laboratories. This paper highlights the research progress, operation categories, clinical application value, domestic standard guidelines, PBRTQC procedure establishment, performance verification, implementation principles, application status and prospects of PBRTQC, so as to promote the recognition, acceptance, reference and wide application of PBRTQC in domestic clinical laboratories.

Important prospects of EV DNA in tumor diagnosis / 中华检验医学杂志

As a new type of intercellular signaling rector, extracellular vesicles (EV) are involved in almost the whole process of tumorigenesis, progression, metastasis, and drug resistance. Therefore, EV have become the ideal biomarker candidates and research hotspots for cancer diagnosis and treatment. However, EV tumor biomarker research mainly focused on RNA and protein, and a small part of the research focused on lipids at the early stage. EV DNA has received little attention and its diagnostic value has gradually been recognized in recent years. Study on the biological characteristics and function of EV DNA may highlight its potential in tumor diagnosis.

Application of extracellular vesicles in tumor diagnosis / 中华检验医学杂志

Extracellular vesicles (EV) are widely distributed, rich in content, and fully participate in the regulation of important cellular physiological activities. As a new technology for tumor diagnosis, the natural advantages of EV include protecting its contents, being easy to obtain from various biofluids, having the potential to be truly non-invasive and high tissue fidelity. In order to transfer EV for clinical use, many isolation technologies such as microfluidics, immunochips, nano-flow cytometry and acoustic capture have been continuously developed. At present, several EV tumor diagnostic products have been applied in the clinical setting. However, due to the great differences in detection methods and platforms, there are still some technical difficulties in its clinical applications. With the accumulation of clinical evidence and the continuous improvement of clinical diagnostic methods, the liquid biopsy based on EV can finally be applied in the clinic.

Artificial intelligence in the laboratory medicine / 中华检验医学杂志

The application of artificial intelligence (AI) technology in laboratory medicine has shown great potential, but it also has many challenges and difficulties. Understanding the basic principles, evaluation methods, application scenarios, advantages and limitations of artificial intelligence can facilitate its use in the working practice. In the near future, AI will be fully applied in clinical testing, enabling laboratory medicine to play a more accurate role in disease diagnosis, curative effect monitoring, prognositic judgment and other aspects. With the help of AI technology, laboratory medicine has a promising future.

How far is the clinical application of EVs-based tumor diagnosis? / 中华检验医学杂志

As a new type of paracrine/autocrine and remote signaling, extracellular vesicles (EVs) involved in almost the whole process of tumorigenesis, progression, metastasis, and drug resistance, which has greatly inspired tumor diagnosis and treatment. EVs have the great potential to be the blood or urine biomarkers and can be used for cancer diagnosis, prognosis and monitoring. Due to its great clinical application value, EVs have become a hotspot in cancer research in recent years. Therefore, in this review, the advantages, progression, and technical challenges of EVs biomarkers in clinical tumors diagnosis are discussed.

Analysis of a pedigree affected with hereditary coagulation factor XII deficiency due to a homozygous 252delAsn deletion of F12 gene / 中华医学遗传学杂志

OBJECTIVE@#To analyze the clinical phenotype and genetic basis of a consanguineous pedigree affected with hereditary coagulation factor XII (FXII) deficiency.@*METHODS@#Following extraction of genomic DNA, all exons and flanking regions of F12 gene were subjected to PCR amplification and Sanger sequencing. ClustalX-2.1-win and MutationTaster software was used to analyze the conservation and impact of the variants on protein function.@*RESULTS@#DNA sequencing showed that the proband carried a homozygous g.6753-6755delACA deletion (p.252delAsn) in exon 9 of the F12 gene, for which her father, mother and brother were heterozygous carriers. The same deletion was not found in her sister.@*CONCLUSION@#The homozygous p.252delAsn deletion probably underlies the hereditary FXII deficiency in this pedigree.

A multicenter research on validation and improvement of the intelligent verification criteria for routine urinalysis / 中华检验医学杂志

Objective:A multi-center and large sample volume study was conducted on the verification and improvement of the early established criteria for intelligent routine urinalysis validation (including the microscopic review rules and manual validation rules, referred to as intelligent criteria for short), in order to improve the clinical application of this intelligent criteria.Methods:A total of 31 456 urine specimens were collected from the inpatients and outpatients in six hospitals in China, from March to September 2019. Firstly, 3105 specimens were analyzed for preliminary verification and improvement of the intelligent criteria based on the results of the microscopic examination and manual validation. Secondly, 28 351 specimens were used to verify the clinical application of the improved intelligent criteria. All samples were manually validated as reference.Results:The approval inconsistency rate of the manual validation rules in the original intelligent criteria was 8.59% (202/2 352), and the interception inconsistency rate was 8.84% (208/2 352). The false negative rate and the microscopic review rate of the microscopic review rules were similar to the previous results. Based on an in-depth analysis of big data and the discussions by senior technicians from eight hospitals, one microscopic review rules and four manual validation rules were added, meanwhile two manual validation rule was deleted. The manual validation standards were unified. Finally, the intelligent criteria was improved. Based on the improved intelligent criteria, for microscopic review rules, the false positive rate, false negative rate (misdiagnosis rate), and microscopic review rate did not change significantly, which were 14.72% (457/3 105), 4.06% (126/3 105), and 24.73% (768/3 105), respectively. The approval inconsistency rate and the interception inconsistency rate of manual validation rules were both reduced to 0; the total manual validation rate of the intelligent criteria was 50.89% (1 580/3 105), and the auto-validation rate was 49.11% (1 525/3 105). The large sample volume verification results were consistent with the preliminary verification results of the improved intelligent criteria.Conclusion:This multi-center and large sample volume study had shown that the improved intelligent criteria had better clinical performance.

How far is the clinical application of EVs-based tumor diagnosis? / 中华检验医学杂志

As a new type of paracrine/autocrine and remote signaling, extracellular vesicles (EVs) involved in almost the whole process of tumorigenesis, progression, metastasis, and drug resistance, which has greatly inspired tumor diagnosis and treatment. EVs have the great potential to be the blood or urine biomarkers and can be used for cancer diagnosis, prognosis and monitoring. Due to its great clinical application value, EVs have become a hotspot in cancer research in recent years. Therefore, in this review, the advantages, progression, and technical challenges of EVs biomarkers in clinical tumors diagnosis are discussed.

Correlation between apolipoprotein E gene polymorphism and lipid metabolism in elderly patients with cardiovascular and cerebrovascular diseases / 中华老年医学杂志

Objective:To investigate the association of apolipoprotein E(ApoE)gene polymorphism with lipid metabolism and common chronic cardiovascular and cerebrovascular diseases in the elderly.Methods:A total of 4 322 elderly outpatients and inpatients from our hospital were enrolled in this retrospective analysis.The distribution of ApoE subtypes in the elderly was analyzed.Then 600 cases(321 cases with atherosclerosis and 279 healthy controls)were included to analyze blood lipid-related biochemical indexes.ApoE genotypes were determined by DNA microarray analysis.Results:ApoE E3 was the most common subtype, making up 67.1%(2 898/4 322)in this study population.The distribution of ApoE subtypes in the elderly with chronic cardiovascular and cerebrovascular diseases was basically the same as that in the elderly population.However, in patients with neurodegenerative diseases, the rate of ApoE E4 carriers was 26.4%(152/576), which was higher than that of ApoE E2 carriers(12.2%, 70/576)( P<0.01). Triglyceride(TG)levels in those with atherosclerosis carrying ApoEε2 and ε4 alleles were higher than those in healthy controls carrying ApoEε2 and ε4 alleles ApoEε2 alleles: (1.85±2.09)mmol/L vs.(2.00±1.44)mmol/L, ApoEε4 alleles: (1.53±1.31)mmol/L vs.(1.84±1.32)mmol/L, P<0.05.TG( OR=4.360, 95% CI: 2.150-8.844), high density lipoprotein cholesterol( OR=0.486, 95% CI: 0.307-0.770), low density lipoprotein cholesterol( OR=2.321, 95% CI: 1.020-5.281)and ApoE alleles( OR=0.335, 95% CI: 0.210-0.533)were risk factors for atherosclerosis( P<0.01). Conclusions:ApoE polymorphism is associated with lipid metabolism in elderly patients.Genetic background may be among the factors affecting lipid metabolism.ApoE ε4 allele carriers have an increased risk of dyslipidemia, cardiovascular and neurodegenerative diseases.

Differential expression of exosomal miRNAs between androgen-dependent prostate cancer cell and androgen-independent prostate cancer cell / 中华检验医学杂志

Objective Studying the differential expression of exosomal miRNAs between androgen-dependent prostate cancer cell and androgen-independent prostate cancer cell,in order to further elucidate the mechanism of androgen-independent prostate cancer and find new targets for its treatment.Methods Prostate cancer androgen-dependent LNCaP cell and prostate cancer androgen-independent LNCaP-AI + F cell (induced by androgen and flutamide) were selected as the study subjects.Illumina HiSeqTM 2500 was used to perform high-throughput sequencing between the two groups.The differentially expressed exosomal miRNAs was verified by quantitative real-time PCR(qRT-PCR).The difference was statistically significant by t-test.Results Through the analysis of high-throughput sequencing results,thirteen molecules were screened increased in extracellular exosomes of the androgen-independent cell,including miR-7-5p,let-7a-5p,miR-375,miR-423-3p,miR-378a-3p,and miR-92a-3p,etc.Among them,miR-7-5p was verified by quantitative real-time PCR to be up-regulated by 19.52-fold (t=9.857,P=0.001).Conclusion Differentially expressed exosomal miRNAs may predict the development of androgen-independent prostate cancer and may further regulate the development of androgen-independent prostate cancer.

The value of methylated circulating tumor DNA as biomarkers in cancer diagnosis and prognosis / 中华检验医学杂志

Circulating tumor DNA (ctDNA) in the blood of cancer patients provides an opportunity for non-invasive sampling of tumor DNA.This "liquid biopsy"allows for detection of copy number variations,tumor-specific mutations,epigenetic changes,and can be used to guide and improve treatment throughout real-time "tracking" the course of tumor disease.Aberrant methylation of specific gene regions can be a very consistent feature of cancer,in contrast to mutations,which makes ctDNA methylation amenable to the design of widely clinical applications in early diagnosis,monitoring disease status,predicting treatment response and predicting prognosis.Therefore,ctDNA methylation detection is considered as one of the most valuable methods for cancer diagnosis and risk assessment.

Optimization for extraction of urine exosomes and effects of freezing on exosomal RNA content / 临床检验杂志

Objective@#To optimize the existing methods of isolation and purification for exosomes from urine and explore the effects of different storage conditions on the content of exosomal RNA in urine. @*Methods@#The exosomes in human urine samples were extracted by different precipitation method, i.e., precipitation following first concentrating and direct precipitation, respectively, and the separation efficiency and cost of the two methods were compared. ExoQuick-TCTM precipitation kit was used to extract exosomes. Nanoparticle tracking analysis technique (NTA) was used to detect the concentration and particle size distribution of exosome. Dynamic light scattering (DLS) was used to detect the potential of exosome. Transmission electron microscopy (TEM) was used to observe morphology of exosomes. western blot was used to analyze the exosomal marker molecules CD63 and Alix. The extraction method of the precipitation following first concentrating was used to verify the reliability of the optimized method in 10 clinical urine samples . Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression levels of exosomal RNA marker let-7c and PSA mRNA in the urinary exosomes from 20 patients with prostate cancer after repeated freeze-thaw (0 [i.e., fresh], 1 , 3 and 5 times) and 9 patients with prostate cancer frozen at -80 ℃ for different time (0 [i.e., fresh], 1, 2 and 4 weeks), and were statistically analyzed by Wilcoxon rank sum test for differences between the 2 groups. @*Results@#The size distribution of exosomes extracted by the two methods was 30 to 150 nm by NTA, both of which were displayed as single peaks. The results of DLS showed that the potentials of exosome extracted by the two methods were negative values. The size of the exosomes extracted by the two methods was consistent observed under TEM namely the diameter distribution was 30 to 150 nm. western blot analysis confirmed that CD63 and Alix, the exosome labeling molecules, existed in the optimized method. The concentration of exosomes extracted from the 10 urine samples all reached 10 9 to 10 11 particles/mL. The contents of let-7c and PSA mRNA in exosomes decreased significantly after 5 freeze-thaw cycles, and the Z values were -1.79 and -1.73, respectively (P＜0.05). The RNA content of the exosomes remained stable after freezing at -80 ℃ for 1 month. @*Conclusion@#The optimized exosome extraction method could reduce greatly the cost under the premises of ensuring the concentration and quality of exosomes. The isolated exosomes may keep stable RNA content after freezing at -80 ℃ for a short time, but could not be frozen and thawed repeatedly for more than 5 times.

A multi-center research on the establishment and validation of autoverification rules for blood analysis / 中华检验医学杂志

Objective@#To establish a set of rules for autoverification of blood analysis, in order to provide a way to validate autoverification rules for different analytical systems, which can ensure the accuracy of test results as well as shorten turnaround time (TAT) of test reports.@*Methods@#A total of 34 629 EDTA-K2 anticoagulated blood samples were collected from multicenter cooperative units including the First Hospital of Jinlin University during January 2017 to November 2017. These samples included: 3 478 cases in Autoverification Establishment Group, including 288 cases for Delta check rules; 5 362 cases in Autoverification Validation Group, including 2 494 cases for Delta check; 25 789 cases in Clinical Application Trial Group. All these samples were analyzed for blood routine tests using Sysmex XN series automatic blood analyzers.Blood smears, staining and microscopic examination were done for each sample; then the clinical information, instrument parameters, test results and microscopic results were summarized; screening and determination of autoverification conditions including parameters and cutoff values were done using statistical analysis. The autoverification rules were input into Sysmex Laboman software and undergone stage Ⅰ validation using simulated data, and stage Ⅱ validation for post-analytical samples successively. True negative, false negative, true positive, false positive, autoverification pass rate and passing accuracy were calculated. Autoverification rules were applied to autoverification blood routine results and missed detection rates were validated, and also data of autoverification pass rate and TAT were obtained.@*Results@#(1)The selected autoverification conditions and cutoff values included 43 rules involving WBC, RBC, PLT, Delta check and abnormal characteristics. (2)Validation of 3 190 cases in Autoverification Establishment Group showed the false negative rate was 1.94%(62/3 190)(P<0.001), autoverification pass rate was 76.74%, passing accuracy was 97.47%; Validation of 2 868 cases in Autoverification Validation Group, the false negative rate was 3.38%(97/2 868)(P=0.002), autoverification pass rate was 42.26%, passing accuracy was 92.00%; Validation of Delta check on 288 cases in Autoverification Establishment Group and 2 494 cases in Autoverification Validation Group showed the false negative rates were respectively 1.39% and 2.61%(P<0.001). (3)Three hospitals adopted these rules of autoverification for 25 789 blood routine samples, and found that the average TAT of blood routine test reports were shortened by 24min, 32min and 7min respectively, the rate of samples reported within 30min were elevated by 33%, 53% and 7%. The autoverification pass rates were 72%-74%.@*Conclusions@#The application of this set of 43 autoverification rules in blood sample analysis can ensure test quality while shortenTAT and improve work efficiency. It is worth pointing out that for the same analytical systems in this research, validation is necessary before application of this set of rules, and periodic validation is required during application to make necessary adjustment; for different analytical systems, as this research provide a way to establish autoverification rules for blood routine tests.Clinical labs may establish their own suitable autoverification rules on the basis of technological parameters. (Chin J Lab Med, 2018, 41: 601-607)

Exosomes: new markers for tumor diagnosis / 中华检验医学杂志

Exosomes are nanoscale extracellular vesicles, which range in size from 30 to 150 nm, and are released under physiological and pathological conditions. The content of exosomes reflects the origin and function of cells. As a form of intercellular vesicular transport, exosome-mediated intercellular communication participates in a variety of physiological and pathological processes. The information transmission mediated by exosomes is associated with pathophysiological status and plays an important role in the pathogenesis of cancer. This review summarizes the research progress of exosomes, including the origin and biological functions, the detection methods, the diagnosis and treatment of tumor.(Chin J Lab Med, 2018, 41: 491-495)

Dilution validation of high level D-dimer samples in Sysmex CS5100 instrument / 国际检验医学杂志

Objective To investigate the necessity of dilution in samples with high concentrations of D-Dimer and optimum dilution multiple.Methods Quality control products and calibration were detected by using Sysmex CS5100 for precision evaluation,including within-batch and between-run precision.Calibration were detected for validation of linear range and clinical reportable.Samples with D-Dimer5 mg/L and FDP>20 μg/mL were also serially diluted and detected to calculated recovery rate.Results Within-batch and between-run coefficients of variation were both less than 3%.Within the scope of 0.207-5.170 mg/L,the linear distribution was fine.The clinical reportable range was 0.207-165.440 mg/L.For samples with D-Dimer5 mg/L and FDP>20 μg/mL,there was obvious antigen excess phenomenon,and gradient dilution was required.Conclusion For samples with D-Dimer>5 mg/L and FDP>20 μg/mL,dilution should be performed to ensure the accuracy of detected results.

Exosome-encapsulated miR-375 in urine as a non-invasive biomarker for prostate cancer diagnosis / 中华检验医学杂志

Objective To analyze the expression of urine exosomal miR-375 in prostate tumors and investigate its clinical utility.Methods A total of 45 patients with PCa,24 with benign prostate hyperplasia (BPH) and 24 healthy individuals were enrolled into this study.Exosomes were isolated from the urine of PCa,BPH and healthy individuals and the total RNA was extracted from the exosomes.The exosomal miR-375 expression was assessed by quantitative real-time PCR and analyzed with the comparative quantification cycle method (2-△△CT).We performed comprehensive biostatistical analyses to explore the clinical value of miR-375 in prostate cancer.Results The urine exosomal miR-375 expression was significantly downregulated in the patients with PCa compared with BPH and the healthy controls (P ＜ 0.01).No statistically significant difference of the urine exosomal miR-375 expression levels between the patients with BPH and healthy individuals was observed (P ＞ 0.05).The urine exosomal miR-375 expression level was also found to be associated with clinical stage and bone metastasis status of the patients with PCa (P ＜0.05),and with the increase of Clinical stage.The expression level of miR-375 decreased.No significant relationship was detected between miR-375 level and the patient's age,gleason score and serum prostate-specific antigen level (P ＞ 0.05).Receiver operator characteristic analyses demonstrated that the urine exosomal miR-375 expression could better differentiate PCa from BPH patients:AUC 0.715 (95％ CI:0.589-0.842) vs PSA AUC 0.632 (95％ CI:0.492-0.771) (P＜0.01).Conclusion The urine exosomal miR-375 could serve as a non-invasive biomarker for the diagnosis of PCa.

Effects of Mycobacterium tuberculosis heparin-binding hemagglutinin(HBHA) on macrophage polar-ization / 中华微生物学和免疫学杂志

Objective To investigate the effects of Mycobacterium tuberculosis heparin-binding he-magglutinin (HBHA) on the polarization of mouse bone marrow-derived macrophages (BMDM) and a mu-rine macrophage cell line(Raw264.7 cells). Methods After HBHA was confirmed to be able to enter into macrophages by immunofluorescence, mouse BMDM and Raw264. 7 cells were treated with LPS (100 ng/ml)+IFN-γ (2.5 ng/ml),IL-4 (20 ng/ml),HBHA (1 μg/ml,3 μg/ml,6 μg/ml,10 μg/ml) and ESAT-6 (5 μg/ml),respectively. IL-6,IL-12 and TNF-α in the supernatants of cell culture were measured by ELISA. RT-PCR was performed to detect the expression of molecular markers of macrophage polarization [inducible nitric oxide synthase (iNOS), TNF-α, Arg-1 and CD206] at mRNA level. Western blot assay was used to detect the expression of iNOS and Arg-1 at protein level. Results Increased secretion of IL-6, IL-12 and TNF-α in the supernatants of cell culture and enhanced expression of iNOS and TNF-α at mRNA level were observed after treating BMDM with Mycobacterium tuberculosis HBHA. Similarly,in HBHA-trea-ted Raw264.7 cells,the secretion of IL-6 and the expression of TNF-α were also up-regulated. Mycobacteri-um tuberculosis HBHA and ESAT-6 had similar effects on murine macrophages. Neither of them could in-crease the expression of Arg-1,a molecular marker of M2 macrophages,in BMDM at protein level,but both enhanced the expression of iNOS,a molecular marker of M1 macrophages,in BMDM and Raw264.7 cells. Conclusion Mycobacterium tuberculosis HBHA can induce the polarization of murine macrophages to M1 phenotype.