Application of capillary electrophoresis in the diagnosis and differential diagnosis of benign and malignant monoclonal gammopathies / 中华检验医学杂志

Objective:To evaluate the value of capillary electrophoresis in the diagnosis and differential diagnosis of benign and malignant monoclonal gammopathies (MGs).Methods:A retrospective analysis of the capillary electrophoresis test results of 2 445 newly diagnosed patients at the Affiliated Hospital of Qingdao University from January 2016 to June 2020 was carried out. Capillary zone electrophoresis and immunosubtractive assay were used to detect serum monoclonal protein (MP). The clinical diagnosis and other information of the patients were collected from the clinical database of the Affiliated Hospital of Qingdao University. Kruskal-Wallis rank sum test was used to compare the different amount of monoclonal protein among multiple groups. Receiver operator characteristic curve (ROC) was used to analyze the diagnostic sensitivity and specificity of the monoclonal protein of each type. Youden index was used to calculate the cut-off values.Results:Among the 2 445 patients, 1 183 were positive for monoclonal protein, of which 944 cases were diagnosed as malignant MG, 174 were monoclonal gammopathy of undetermined significance (MGUS), and 65 cases were monoclonal gammopathy of renal significance (MGRS). The percentages of M protein types from high to low is immunoglobulin G(IgG)-κ, IgG-λ, IgA-λ, IgA-κ, free λ light chain, free κ light chain, IgM-κ, double clone, and IgM-λ. The levels of MP of IgG, IgA, IgM and FLC in the malignant MG group were all higher than those in the MGUS group, with statistical significance( P<0.01). The MP levels of IgG and IgA types in malignant MG group were higher than that in MGRS group ( P<0.01). ROC curve analysis showed that the MP of IgG, IgA, IgM and FLC types had good diagnostic efficacy for malignant MG ( P<0.01), and their AUC values were 0.947 (95 %CI 0.926-0.968), 0.930 (95 %CI 0.895-0.966), 0.844 (95 %CI 0.722-0.967) and 0.865 (95 %CI 0.781-0.950), respectively. For IgG, the cut-off value was 14.24 g/L, and the diagnostic sensitivity and specificity were 88.5% and 90.1%, respectively. The cut-off value of IgA was 8.88 g/L, and the sensitivity and specificity of IgA were 87.9% and 81.4%, respectively. For IgM, the cut-off value was 26.93 g/L, and the sensitivity was 64.4% and the specificity was 90.9%. For FLC, the cut-off value, diagnostic sensitivity, and specificity was 7.08 g/L, 85.9%, and 77.8%, respectively. Conclusions:Capillary electrophoresis immunotyping technique can be used to diagnose malignant MG such as multiple myeloma (MM) and non-Hodgkin′s lymphoma (NHL), as well as to screen and track diseases like MGUS and MGRS. Serum MP level can be used to distinguish malignant MG from benign MG effectively.

The application of modified capillary immunotyping technique for cryoglobulin identification / 中华检验医学杂志

Objective To establish and evaluate the application of modified capillary immunotyping for cryoglobulin qualification .Methods Referred to literature and benchwork experience , a modified capillary immunotyping technique was set up for cryoglobulin identification . Seventy-eight cryoglobulin positive specimens were collected by a standard method in Peking Union Medical College Hospital from November 2016 to July 2017.Thirty-nine samples were identified the type of the cryoglobulin simultaneously by modified capillary immunotyping ( CI ) and immunofixation electrophoresis ( IFE ) .Results Using the modified capillary immunotyping method , the types of cryoglobulin in seventy-eight specimens were identified.The number of cases decreased in the order of Ⅲ, Ⅱ and Ⅰ type of cryoglobulin .The clinical characteristics coincidence with previous reports .The modified CI method had a dramatic advantage in the speed, clarity, and accuracy of results compared with IFE .The ratio of reportable cases between these two methods was 1:0.87.Conclusion The modified capillary immunotyping was an accurate and easy method for cryoglobulin qualification , and feasible for clinical application .

The application of IFCC reference methods in methodological evaluation of glycated hemoglobin measurement systems / 中华检验医学杂志

Objective To perform a methodological evaluation study of 4 HPLC based systems and a capillary electrophoresis based system , with the International Federation of Clinical Chemistry and Laboratory Medicine ( IFCC) glycated hemoglobin reference method as a comparative method .Methods 40 hemolysis samples of variety concentrations were prepared .The samples were measured by IFCC reference method and 5 glycated hemoglobin testing systems , respectively and trueness verification was performed according to the Clinical &Laboratory Standards Institute (CLSI) guideline EP9-A3.Whole blood samples were used to test the systems'precision, linearity and analytical interferences .Results The average CV of IFCC reference method results of 40 hemolysis samples was 1.4％( range from 0.2％to 2.5％).No outlier was found in the results of the 5 testing system.The slopes ranged from 0.9902 to 1.0267, and intercepts from -0.1526 mmol/mol to +0.1512 mmol/mol, squared correlation coefficient from 0.9962-0.9971, biases at two medical decision level were less than 0.3％HbA1c.Within-laboratory precisions were less than 2％ (NGSP unit).Bias between all test results and predicted results were less than 5％.High concentration of glucose showed certain interference to glycated Hemoglobin tests but had little influence in clinical practice.Conclusions The results of the 5 testing system are comparable to the IFCC reference method , the results of precision and linearity evaluation meet the requirements of related guidelines .

Measurement of effective constituents contents in rhubarb by capillary electrophoresis / 重庆医学

Objective To adopt the capillary electrophoresis(HPCE) for detecting the contents of emodin,chrysophanol and rhein in the extract of rheum officinale.Methods The detection was executed with a fused-silica capillary column(67.4 cm× 75.0 μm,effective length 51.0cm) as the separation column.The buffer solution consisted of 60 mmol/L Na2B4O7,40 mmol/L Na2CO3 and 40 mmol β-cyclodextrin(pH 8.9).The detection wavelength was 254 nm.Results RSD of precision in emodin,chrysophanol and rhein was 1.79％,4.46％ and 2.30％.respectively The linear range of 3 components was 1.1-19.0 mg/mL;the average recovery rates of emodin,chrysophanol and rhein were 100.14 ％,99.65 ％ and 98.44 ％ respectively,RSD was 2.43 ％,2.54 ％ and 2.02％ respectively(n=3).Conclusion The method can be used in the content determination of rheum officinale extract.

The application of GeXP based multiplex RT-PCR assay for respiratory viral infection in children / 中华检验医学杂志

Objective To detect twenty types/subtypes of respiratory viruses with the GeXP (genomelab genetic analysis system) based mRT-PCR (multiplex reverse transcription-polymerase chain reaction) assay,and discuss its value in pediatric respiratory viral infection.Methods A retrospective study was conducted on a total of 1 305 inpatient children (0-6 years old) admitted to the Department of Respiratory in Children's Hospital of Hebei Province from May 2014 to November 2014.The sputum samples were collected and the viral DNA or RNA was extracted.After examining the specificity and sensitivity,the GeXP-based mRT-PCR assay was performed to test 20 types/subtypes of respiratory viruses.The obtained results were evaluated in comparison with those of fluorescence real-time PCR and direct sequencing.Chisquare test was conducted by SPSS16.0.Results Among 1 305 hospitalized children,the positive rate of respiratory virus was 58.31％ (761/1 305).The positive rate of respiratory syncytial virus (RSV,17.32％,226/1 305) was the highest followed by the parainfluenza virus 3 (PIV-3,16.02％,209/ 1 305),human rhinovirus (HRV,11.95％,156/1 305),adenovirus (ADV,5.06％,66/1 305),human bocavirus (HBoV,3.14％,41/1 305) and the parainfluenza virus 1 (PIV-1,2.07％,27/1 305).Multiple virus infection was found in 92 children (7.05％).The highest positive rate of respiratory virus was observed in 1-2 years old children (83.28％,249/299);the lowest rate was found in 5-6 years old children (37.68％,26/69).Conclusions The proposed GeXP-based mRT-PCR assay possessed the advantage of high throughput,sensitivity,specificity and rapidity in the detection of twenty types/subtypes of respiratory viruses.It provided a thorough investigation of viral pathogens involved in acute respiratory infection thus could satisfy the clinic requirement and provide epidemic data on the disease prevention.

Determination of trace oxalate and citrate in urine with capillary electrophoresis / 中华检验医学杂志

Objective To establish a method for determining oxalate and citrate in urine simultaneously by capillary electrophoresis.The components,the concentration and pH of the buffer solution,the separation voltage and the injection time on theseparation were studied in detail.Methods The separations were carried out using potassium dihydrogen phosphatebuffer ina fused-silica capillary tubeby capillary zone electrophoresis (CZE) and the detection were monitored by UV.24 h-urine samples from patients (n =5) and health control (n =5) were collected from Zhongshan Hospital of Fudan University for systematically validating the method developed.Results The optimized separations were carried out using a 50 mmol/L potassium dihydrogen phosphatebuffer solution (pH 6.5) in a fused-silica capillary tube of 50 cm × 50 μm I.D.Injections were made by using the pressure mode for 10 s at 34 mbar.The detections were monitored by a UV at 200 nm after samples were separated at avohage of 30 kV.Under the seconditions,urinary oxalate and citrate were separated completely within 5 min.The relative standard deviations of migration time and peak area within-run foroxalate and citrate were less than 1％ and 3.0％ and the betweenrun relative standard deviations were less than 2.0％ and 4.0％,respectively.The detection limits were 1 mg/L for both oxalate and citrate.The linearity ranges of oxalate and citrate were both 0-500 mg/L with the correlation coefficient between 0.999 5 and 0.995 4 (P ＜ 0.05),respectively.The average recoveries were 102.38％ for oxalate and 92.74％ for citrate.Conclusion This method is proved to be simple,sensitive and accurate,and also applied to determine oxalate and citrate in urine samples with satisfactory results.

Simultaneous detection of paraquat and creatinine in urine by capillary electrophoresis / 中华检验医学杂志

Objective To establish a method for detecting the concentration of paraquat (PQ) and creatinine(Cr) in urine simultaneously by capillary electrophoresis.Methods Experimental methodological study.8 acute PQ poisoned patients who were treated in the First People's Hospital of Lianyungang from January 2011 to June 2012 were collected.2 were male,and 6 were female.The separation were carried out using a 25 mmol/L pH1.97 glycine-HCl buffer(containing 40 mmol/L NaCl) in a fused-sillica capillary tube of 47 cm ×75μm I.D.by capillary zone electrophoresis.Urine had been injected by pressure for 4 s after samples were centrifuged and diluted for 10 times with H2O.The detection were monitored by a diode-array detector at 200 nm while samples were separated at a voltage of 20 kV.A systemic methodological evaluation of this method was carried out (The linear range,detection limit,repeatability test,recovery test and interference test).And the method was used to detect the concentration of PQ and Cr in PQ poisoned patients' urine.Results The peaks of PQ and Cr appeared within 5 min.The linear ranges of PQ and Cr were 2-1000,10-5000 μmol/L,respectively,with the correlation coefficients of 0.9997 and 0.9999 (P ＜0.01).The detection limits were 1.0 μmol/L for PQ and 5.0 μmol/L for Cr.The mean within-day(n =10) CVs of peak area for PQ and Cr were 2.84％ and 1.72％,while the mean inter-day(n =10) CVs of peak area were 3.62％ and 3.06％.The average recovery rate of PQ and Cr were 88.6％ and 90.2％ respectively.Diquat(DQ) didn't interfere with the assay.The range of PQ/Cr(μmol/mmol) for 8 cases was 8.9-2215.Conclusions A method was established successfully for the rapid determination of PQ and Cr in urine by capillary electrophoresis.The CE method devised here for direct measurement of urinary PQ and Cr from PQ poisoned patients is simple,fast,automatic and with good repeatability.It is an ideal method for rapid detection of urinary PQ in PQ poisoned patients.

Evaluation of major methods of hemoglobin analysis / 中华检验医学杂志

Hemoglobinopathy is a kind of hereditary hematonosis caused by the disproportion among normal hemoglobins or formation of abnormal hemoglobins.Hemoglobin analysis plays an important role in screening and diagnosing hemoglobinopathy.Emerging methods have been applied continuously to hemoglobinometry since the early 20th century.Nowadays,agarose gel electrophoresis,high performance liquid chromatography and capillary electrophoresis are widely used with mass spectrometry as the latest applied method.All these methods have their own features.Appropriate combination of these methods will lead to more scientific and reliable results.

Detection of TRAPPC2 gene mutation in a Chinese pedigree of X-linked spondyloepiphyseal dysplasia tarda / 中华检验医学杂志

Objective To identify the mutation of trafficking protein particle complex 2 (TRAPPC2) gene in a large Chinese pedigree with X-linked spondyloepiphyseal dysplasia tarda by the PCR-based capillary electrophoresis methods.Methods The blood samples were collected from a large Chinese pedigree of three generations with six affected persons with X-SEDT.Four exons comprising the TRAPPC2 gene open reading frame as well as their exor/intron boundaries were analyzed by argrose electrophoresis and bidirectional direct sequencing of PCR products.Fluorescence labeled fragment analysis was performed by capillary electrophoresis.Results A 5-bp deletion mutation of TRAPPC2 gene in exon 5,c.262_266delGACAT (D88del; I89fX12),was identified in the proband and his unaffected mother(a heterozygote) in the Chinese family with X-SEDT,but no other sequence change occurring in exons 3,4 and 6 was detected.The old sister of proband was determined being carriers because she carries the deletion fragment allele of exon 5 PCR product and the young sister being normal individuals because she carries the wild allele of TRAPPC2 gene.Conclusions The mutation c.262_266delGACAT (D88del; I89fX12) of TRAPPC2 gene was firstly reported in Chinese people.The mutation of c.262_266delGACAT (D88del; I89fX12) in TRAPPC2 gene may be the pathologic cause of the patients in the X-SEDT pedigree.Fragment analysis combined with DNA sequencing by capillary electrophoresis method is effective laboratory test in the small deletion mutation analysis and carriers screening in X-SEDT family.

Avaliação de estratégia combinada de eletroforese capilar e PCR triplo metilação-específica para diagnóstico molecular da Síndrome do X Frágil / Assessment of a combined strategy of capillary electrophoresis and methylation-specific triple polymerase chain reaction for molecular diagnosis of the Fragile X Syndrome

Introdução: a síndrome do X frágil é a principal causa de retardo mental de natureza familiar. Suas características clínicas pouco marcantes fazem com que um diagnóstico de certeza a partir de testes moleculares seja imprescindível. Objetivo: descrever e avaliar as vantagens e desvantagens de uma estratégia combinada de PCR triplo metilação-específica e eletroforese capilar para o diagnóstico molecular da síndrome do X frágil, visando baixo custo, exequibilidade, reprodutibilidade e sensibilidade. Métodos: foram coletadas 43 amostras de sanguede pacientes com déficit cognitivo e de suas mães, quando indicado. Tais indivíduos possuíam exame citogenético positivo, fenótipo e/ou história familiar sugestivas de síndrome do X frágil. Após extração de DNA, foi realizada eletroforese capilar com marcadores fluorescentes,tratamento com bissulfito de sódio e três reações de PCR metilação-específicas em cada amostra. Resultados: foi possível determinar o genótipo em 29 pacientes: 23 (14 homens e nove mulheres) apresentavam alelos com tamanho normal e seis (todos do sexo masculino) possuíam alelos na faixa de mutação completa. Em outras seis amostras, todas do sexo feminino, foi possível determinar um alelo na faixa normal e outro alelo alterado, entretanto, sem diferenciação entre faixa de pré-mutação ou de mutação completa. Nas demais oito amostras(cinco homens e três mulheres), não se pôde determinar o genótipo. Conclusões: a técnica proposta faz uma triagem de pacientes, mas apresenta desvantagens, como não terem sido obtidos resultados satisfatórios com as reações para alelos metilados e a análise dos rastrosdas PCRs com três primers ter se mostrado difícil e dependente de observador.

Introduction: The fragile X syndrome is the main cause of inherited mental retardation. Its very small remarkable clinical characteristics make that a surely diagnosis from molecular tests be indispensable. Objective: To describe and assess the advantages and disadvantages of a combined strategy of methylation-specific triple polymerase chain reaction (PCR)and capillary electrophoresis for the molecular diagnosis of the fragile X syndrome, seeking low cost, feasibility, reproducibility, and sensibility. Methods: 43 blood samples were collected from patients with cognitive deficit and their mothers, when indicated. Such subjects presented a positive cytogenetic exam, phenotype and/or familiar history suggestive of the fragile X syndrome. After DNA extraction, capillary electrophoresis with fluorescent markers, treatment with sodium bisulfite, and three methylation-specific PCR reactions were performed in each sample. Results: It was possible to determine the genotype in 29 patients: 23 (14 men and 9 women) presented alleles with normal size and six (all male) had them in the complete mutation range. In other six female samples, it was possible to determine an allele in the normal range and another altered; nevertheless, without differentiation between the pre-mutation or complete mutation ranges. In the other eight samples (five men and threewomen), it was not possible to establish the genotype. Conclusions: The suggested technique does a patient?s screening, but it has some disadvantages such as non-satisfying results been seen with the reactions for methylated alleles and analysis of the PCRs tracks with three primers being difficult and dependent on the observer.

Detection of NPM1 gene mutation in acute myeloid leukemia / 中华检验医学杂志

Objective To analyze the frequency of NPM1 mutation in de novo acute myeloid leukemia (AML) patients and the relationship between NPM1 mutation and chromosome alterations,as well as FAB subgroups,and to analyze the mutation type.MethodsA total of 99 de novo AML patients from 2004 to 2010 in China-Japan Friendship Hospital were studied.Genomic DNA was amplified by polymerase chain reaction (PCR),denaturing polyacrylamide gel electrophoresis (PAGE) and capillary electrophoresis were used to detect the mutation of NPM1 gene in 99 AML patients,and karyotyping was performed in 72 AML patients by G banding techniques.DNA sequences analysis of NPM1 mutation was performed on 10 patients.Chi-square test was used to compare the frequencies of NPM1 mutation among the different subgroups,and McNemar's test was used to compare the different rates between denaturing PAGE and capillary electrophoresis.ResultsThe frequencies of NPM1 mutations were detected in 15％ (15/99) of AML patients with capillary electrophoresis and 11％ (11/99 ) with denaturing PAGE(x2 =2.25,P ＞0.05 ).The NPM1 was at different rates in M2(27％,8/30),M5(32％,6/19),M6( 13％,1/8),respectively (x2 =1.06,P ＞ 0.05 ),and not detected in the other subgroups.NPM1 mutation in patients with normal karyotype(26％ ) was more prevalent than patients with abnormal karyotype (4％) (x2 =5.61,P ＜ 0.05)All of the 10 patients were of A type ( c.860_863dupTCTG).The C-terminal portion of the NPM protein by replacing the last seven amino acids(WQWRKSL) with 11 residues (CLAVEEVSLRK).Two intronic deletions were novel,one case was IVS10-18_-15delCTTT,the other was IVS10-17_-15delTTT.Conclusions NPM1 mutations represents a common genetic abnormality in AML patients,and NPM1 mutation in patients with normal karyotype is higher than patients with abnormal karyotype.Two new intronic deletion mutations are identified.

Detection Limit of Monoclonal B-Cells Using Multiplex PCR and Laser-Induced Fluorescence Capillary Electrophoresis

BACKGROUND: The identification of monoclonality has been widely used for making diagnoses of lymphoproliferative lesions. Awareness of the sensitivity and detection limit of the technique used would be important for the data to be convincing. METHODS: We investigated the minimum requirement of cells and sensitivity of gel electrophoresis (GE) and laser-induced fluorescence capillary electrophoresis (LFCE) for identifying IgH gene rearrangement using BIOMED-2 protocols. DNA extracted from Raji cells were diluted serially with peripheral blood mononuclear cells (PBMNCs) DNA. DNA from mixtures of diffuse large B-cell lymphoma (DLBCL) and reactive lymph nodes were also serially diluted. RESULTS: For Raji cells, the detection limit was 62 and 16 cell-equivalents for GE and LFCE, respectively. In the condition with PBMNCs mixture, 2.5% and 1.25% of clonal cells was the minimum requirement for GE and LFCE, respectively. In 23% of DLBCL cells in tissue section, the detection limit was 120 and 12 cell-equivalents for GE and LFCE, respectively. In 3.2% of DLBCL cells, that was 1,200 and 120 cell-equivalents for GE and LFCE, respectively. CONCLUSIONS: These results show that LFCE method is more sensitive than GE and the sensitivity of clonality detection can be influenced by the amount of admixed normal lymphoid cells.

Expression of N-glycome in sera of patients with hepatocellular carcinoma / 第二军医大学学报

Objective: To compare the expression profiles of serum glycomics between hepatocellular carcinoma(HCC) patients and normal adults, in an attempt to search for new biomarkers for HCC. Methods: Serum samples were obtained from HCC patients with hepatitis B virus(HBV) infection (n = 100) and normal adults(n = 130). Profiles of serum N-glycome were determined by capillary electrophoresis (CE) based on DNA sequencing equipment. Results: Each sample had a profile with 11 peaks; the peaks were adjusted and quantified. Peaks 1, 2, 9, and 11 in HCC group were higher than those in the control group; peaks 6, 8 and 10 were lower in HCC group than those in control group (P<0.05). The changes of peak values of several peaks were correlated with tumor markers such as alpha-fetoprotein(AFP), HBV DNA copies, albumin and prothrombin time. Conclusion: N-glycome profiles in serum are significantly different between HCC patients and normal adults, which may provide evidence for clinical diagnosis of HCC.

Determination of a peptide in urine of patients after allogeneic hematopoietic stem cell transplantation by LIF-CE-IA / 中华检验医学杂志

Objective To develop an assay with polyclonal antibodies against a fragment derived from human albumin for determination of a peptide in urine, and to provide an early diagnostic tool for GVHD. Methods A small peptide composed of 16 amino acids (LVRYTKKVPQVSTPTL) was synthesized artificially. The immunogen was then diluted into 100 μg/kg body weight of rabbit. Subcutaneous injection in the immune animals was performed on both sides of spine and groin with 2.5 ml antigen suspension for three times, in order to prepare the polyclonal antibodies. The peptide antigen was then labeled with fluorescein isothiocyanate (FITC), and detected by LIF-CE-IA with the pre-prepared antibodies. Results The titer of serum. The migration time of the labeled antigen was 5.93 min, while the migration time of antigen-antibody complex was 6.47 min. The linear range of the method was 16 to 512 ng/ml, and the minimum detection limit was 10 ng/ml. Conclusions The polyclonal antibodies against the peptide antigen was isolated successfully, which possessed high titer and specificity. These results indicated that the assay was simple and rapid and could be applied for the early diagnosis of patient with GVHD.

La electroforesis capilar como una nueva estrategia en la medicina y el diagnóstico clínico / Capillary electrophoresis, a new diagnostic tool

Capillary electrophoresis (CE) may replace many conventional clinical laboratory methods, such as electrophoresis, Southern blotting, sequencing and HPLC (High-performance liquid chromatography). It is an ideal technique due to analytical speed, the possibility of handling great amount of samples, its capacity to separate small molecules according to their size, charge, hydrophobic and stereo-specificity its good reproducibility the use of small amounts of sample and reagents, its low costs and easy handling. The diagnosis of hereditary diseases or the predisposition to polygenic diseases related to specific mutations or polymorphisms can be carried out with this method. In clinical laboratories, this technique is being used for the analysis of several substrates present in urine or serum and for the diagnosis of some infectious agents. It is also a firsthand tool in forensic medicine for human identification and anthropology.

Comparison of the clinical application of different methods for detection of NPM1 gene mutations in leukemia / 中华检验医学杂志

Objective To analyze nucleophosmin (NPM1) gene mutations in exon 12 in patients with acute myeloid leukemia (AML) and evaluate the clinical appliance of three methods which are frequently used for detecting gene mutation. Methods Genomic DNA from bone marrow of 54 AML patients was detected by PCR for NPM1 exon 12 and screened by PCR-capillary electrophoresis, denature high-performance liquid chromatography (DHPLC) and direct sequencing separately. FLT3-ITD (FMS-like tyrosine kinease internal tandem duplication) was detected by agarose gel electrophoresis and PCR-capillary electrophoresis. Results Seven AML sample harbored NPM1 gene mutations. Five of them were the most common mutation, known as type A (an insertion of a TCTG tetranucleotide at position 960 bp). One of them was type D (an insertion of a CCTG tetranuclectide at position 960 bp). The new variant was a deletion of a TGGCAGTG sequence at 958 bp and insertion of a GCCCGCGGTTTA sequence instead. The detection ratio of the three methods was all 100% and capillary electrophoresis was more rapid, reliable and easier than the other two methods. Moreover it could detect FLT3-ITD simultaneously. The resolving power of DHPLC was affected by many factors. The direct sequencing method was tedious and the heterozygous sequence might be misread. Conclusions There is a new mutation at position 958 bp with a 12-nucleotide insertion and substitution. PCR-capillary electrophoresis is convenient to screen NPM1 mutations of AML in clinical practice.

Establishment and application of medical method for quantification of genomic DNA methylation in methotrexate drug resistance cells / 中华检验医学杂志

Objective To establish a rapid and convenient method for determination of genomic DNA methylation in cells.Methods Five standard substances (dC, mdC, dA, dT and dG) were separated by high-performance capillary electrophoresis.Bare fused-silica capillary was used and eletrophoresis buffer was 48 mmol/L NaHCO3 with 60 mmol/L SDS, pH 9.6.The temperature of separation was controlled at 25 ℃ and a voltage of 20 kV was applied.The separation of the mixture was performed at a wavelength of 256 nm with UV-Vis detection and injection time was 5 seconds at 0.7 psi.Under optimal condition,genomic DNA methylation in methotrexate drug-resistant A549 cells was detected.Results The optimal condition was made by adjusting SDS concentration(40, 60, 80 mmol/L), pH value of running buffer(9.4,9.6, 9.8), voltage(15, 17, 19, 20, 22 kV), injection time(5, 10, 15, 20, 30 s) and capillary temperature(15, 20, 25, 30 ℃).The method for determination of genomic DNA methylation in cells was established.Five substances were completely separated by high-performance capillary electrophoresis in 10 mins.Intra-day coefficient of variation was less than 0.2% and inter-day coefficient of variation was less than 2%.The minimal detection limit was 2 μmol/L.Percentage of mdC in A549 parent cells was (4.80 ±0.52) %.Percentage of mdC in 15, 30, 40 μmol/L methotrexate drug-resistant A549 cells were (4.20±0.44) %, ( 3.70 ± 0.36 ) %, (3.10±0.35 ) %, respectively.Conclusions Genomic DNA methylation can be quantificated by high-performance capillary electrophoresis efficiently, rapidly, conveniently and sensitively.Genomic DNA methylation in methotrexate drug resistance cells decreases significantly.

The quantitative assay and clinical significance of JAK2V617F mutation in 131 patients with chronic myeloproliferative disorders / 中华内科杂志

Objective To investigate the frequency and mutational status of JAK2V617F mutation in Chinese patients with chronic myeloproliferative disorders(CMPD) and to study the relative quantification of mutated JAK2 mRNA and the clinical significance. Methods JAK2V617F mutation and the mutational status were screened with amplification-refractory mutation system polymerase chain reaction(ARMS-PCR), the relative quantification of mutated JAK2 mRNA was studied by using capillary electrophoresis. Results A higher prevalence of JAK2V617F in either the heterozygotc or homozyote status in essential thrombocythemia (ET) was observed in elderly patients with ET (P<0.05). The presence of JAK2V617F was found to be significantly correlated with the age at diagnosis (P<0.05); patients with age ≥ 60 years showed significantly higher JAK2 mutated RNA levels than those with age < 60 years (P<0.05); the presence of JAK2V617F in polycythemia vera (PV) and ET was found to be significantly associated with higher hemoglobin level and higher leukocyte count (P< 0.05). In addition, higher leukocyte count was observed in homozygous ET patients than in heterozygous ET patients (P<0.05). The frequency of JAK2V617F mutation and the prevalence of homozygote in PV patients were higher than those in ET patients (P<0.05). The differences of JAK2V617F mRNA levels among PV, ET and chronic idiopathic myelofibrosis (IMF) were not significant. Conclusions ARMS-PCR technique can be used to detect the frequency and mutational status of JAK2V617F mutation owing to its sensitivity and along with capillary electrophoresis, quantitative assay for mutated JAK2 mRNA, diagnosis of CMPD and judgement of prognosis become possible.

Detection of folylpolyglutamate synthetase in methotrexate enantiomer induced resistant A549 cells by capillary electrophoresis-laser-induced fluorescence / 中华检验医学杂志

Objective To establish a method for detection folylpolyglutamate syntbetase (FPGS),explore the change of FPGS in the drug-resistant A549 cells induced by methotrexate(MTX) enantiomer,and provide new tools to further investigate drug resistant mechanism. Methods A549 cell lines induced by L-( + )-MTX and D-( - )-MTX (25 μmol/L) were chosen to raise three cell lines as compared with MTX-sensitive cell line. Then FPGS were extracted for the CEIA-LIF and western blot was performed. After validation, FPGS antibodies were labeled by fluoreacein isothiocyanate (FITC) and produced a immune response with former-extracted FPGS. CELA-LIF can separate and detect labeled proteins according ruination time of the protein with different size and detect FPGS in drug resistant cell lines induced with L-(+)-MTX and D-(- )-MTX. The accuracy was evaluated as compared with western blot assay. Results The separation time of CEIA-LIF for labeled FPGS antibody and the immune complexes were7. 1 min and 8.9 min, and the resolving power was 4. 5. The process of protein separation and detection can be accomplished in less than 10 minutes. Western blot analysis showed there was no non-specific bands appears in the extract of these three cell lines after the freeze-thaw in liquid nitrogen. The minimum detection level in sensitive cell strains was 0. 68 mg/μl. The consent of FPGS in L-(+)-MTX and D-( - )-MTX induced cells were 46. 59% and 48. 36% compared with drug sensitive cell strains with CELA-LIF. Conclusions CELA-LIF was established in this experiment. It is efficient and sensitive for detecting of FPGS, which is similar to western blot method. The level of FPGS in L-( + )-MTX and D-( - )-MTX induced drug resistant cell lines is significantly lower, indicating the expression of FPGS is damaged.

Screening of active saccharides interacted with granulocyte colony-stimulating factor using capillary electrophoresis and their effects on leukemia cells / 中华检验医学杂志

Objective To screen out the bioactive sulfate saccharides interacted with granulocyte colony-stimulating factor (G-CSF) and provide some scientific data on their pharmacology. Methods The interactions between G-CSF and 15 sulfate saccharides with various chain lengths and extent of sulfation were studied by capillary electrophoresis. Furthermore, the effects of these G-CSF binding saccharides on the proliferation and differentiation of leukemia cell line and NFS-60 were also investigated. Results Except for the hepatosaccharide, degradable carrageenan, Dextran sulfate (DexS) with a low molecular mass the nonsulfated sancharide and alkaline chitosan oligosaccharide, the rest of saccharides had interact with G-CSF.DexS500, λ carrageenan, ι carrageenan, κ carrageenan and low molecular weight heparin-1(LMWH-1)with anticoagulant activity could inhibit the NFS-60 growth. Compared with the absorbency of the control group (100.00±3.00) after these saccharides interacted with the NPS-60 cells, their relative absorbencies were 31.33±2.08,37.67±2.08,45.00±1.20,43.00±2.65 and 74.67±1.52, respectively. Their differences had a statistical significance(t=27.94,26.71,26.42,26.61,10.54,P<0.01). The morphological analytic results showed that the LMWH and lambda carrageenan as well as DexS with a molecular mass of 500 000 induce effectively promyclocyte to transit metamyelocyte. Flow cytometry results showed enhanced expressions of CD11b, CD18 and CD45 after treatment with the LMWH, lambda carrageenan and DexS500,respectively. Cell cycle analysis indicated that the S phase population decreased after treatment with these saccharides. Conclusions Capillary electrophoresis is an effective medicine-screening technique that is used to screen some of these saccharides which could bind to G-CSF. And it is found that the interactions are correlated with the chain length, the extent and the position of sulfation of these saccharides.