Analysis of HbA1c on an automated multicapillary zone electrophoresis system

Hemoglobin A1c (HbA1c) is a frequently requested laboratory test and there is thus a need for high throughput instruments for this assay. We evaluated a new automated multicapillary zone electrophoresis instrument (Capillarys 3 Tera, Sebia, Lisses, France) for analysis of HbA1c in venous samples. Routine requested HbA1c samples were analyzed immunologically on a Roche c6000 instrument (n?=?142) and then with the Capillarys 3 Tera instrument. The Capillarys 3 Tera instrument performed approximately 70 HbA1c tests/hour. There was a strong linear correlation between Capillarys 3 Tera and Roche Tina-Quant HbA1c Gen 3 assay (y?=?1.003x – 0.3246 R^2?=?.996). The total CV for the 12 capillaries varied between 0.8 and 2.2% and there was a good agreement between duplicate samples (R^2?=?.997). In conclusion, the Capillarys 3 Tera instrument has a high assay capacity for HbA1c. It has a good precision and agreement with the Roche Tina-Quant HbA1c method and is well suited for high volume testing of HbA1c.     

Importance of HPLC confirmation of problematic carbohydrate-deficient transferrin (CDT) results from a multicapillary electrophoresis routine method

BackgroundThis study presents results from a routine laboratory setup for carbohydrate-deficient transferrin (CDT) measurement, using initial capillary electrophoresis (CE) analysis and, in problematic cases, further investigation by high-performance liquid chromatography (HPLC).MethodsCDT by the Capillarys CDT assay (disialo- and asialotransferrin) was measured on a Capillarys 2 multicapillary CE system (Sebia). In cases of abnormal peak profiles or other interferences in the electropherogram, samples were remeasured following sample cleanup by “Sample treatment” (Sebia). If the problems remained, or otherwise when confirmatory testing was considered important, samples were analysed by an HPLC candidate reference method.ResultsIn about 0.6% of the routine specimens, reanalysis was performed after sample cleanup. Confirmatory HPLC analysis was needed in half of the cases. Quantification problems were observed for genetic transferrin variants and samples showing a variety of abnormal peak profiles. In these cases, HPLC analysis provided accurate quantification, or at least an estimation (positive/negative), of the CDT level. Confirmatory HPLC analysis was also important for evaluation of borderline values, because only part of those cases were indicated to be true positives.ConclusionsThese results indicated that a routine laboratory setup for CDT measurement, using initial high-throughput multicapillary CE analysis with a confirmatory HPLC analysis option, will combine rapid quantitative and qualitative workflow with enhanced patient safety.     

全自动多通道毛细管区带电泳技术在血红蛋白分析中的临床应用

目的探讨全自动多通道毛细管区带电泳技术在血红蛋白分析中的临床应用价值。方法采用法国SebiaCapillarys2电泳系统（Version5．50）及配套试剂,全血样品经系统自动处理后,在9．8kV电压、34℃、pH9．4的缓冲液条件下,在8条并联的毛细管（17．5cm×25μm）内进行血红蛋白电泳,415nm下直接检测各组分的百分比浓度。结果分析183例健康成人血红蛋白电泳结果,确定本实验室参考范围HbA2：2．1％～3．2％。应用HbAFSC质控进行精密度试验,各组分的cV值在（批内：0．21％～1．07％;批间：0．60％43．10％）之间。检测6045例临床需要进行溶血性黄疸诊断和鉴别诊断的患者标本,检出HbA2增高379例;HbF增高203例;HbH带27例;HbE带8例;HbS带4例;HbJ—K带3例;HbBart’s2例;HbD1例。结论SebiaCapillarys2电泳系统能够清晰分辨HbA2、HbF增高及其他异常区带;可准确地将HbC和HbE与HbA2区分;还可完美区分和聚焦HbA与HbS之间的HbF;并可以轻易区分移动速度相近的HbS和HbD2具有自动化程度高、操作简便、快速准确的特点,适合医疗机构临床实验室在血红蛋白分析中常规应用。     

Analysis of inflammatory response in human plasma samples by an automated multicapillary electrophoresis system.

A new automated multicapillary zone electrophoresis instrument with a new high-resolution (HR) buffer (Capillarys with HR buffer) for analysis of human plasma proteins was evaluated. Albumin, alpha(1)-antitrypsin, alpha(1)-acid glycoprotein, haptoglobin, fibrinogen, immunoglobulin (Ig)A, IgG and IgM were determined nephelometrically in 200 patient plasma samples. The same samples were then analyzed on the Capillarys system (Sebia, Paris, France). The albumin concentration from the nephelometric determination was used for quantification of the individual peaks in the capillary electrophoresis (CE) electropherogram. There was strong linear correlation between the nephelometric and electrophoretic determination of alpha(1)-antitrypsin (R(2) = 0.906), alpha(1)-acid glycoprotein (R(2) =0.894) and haptoglobin (R(2) = 0.913). There was also good correlation between the two determinations of gamma-globulins (R(2) = 0.883), while the correlation was weaker for fibrinogen (R(2) = 0.377). The Capillarys instrument is a reliable system for plasma protein analysis, combining the advantages of full automation, good analytical performance and high throughput. The HR buffer in combination with albumin quantification allows the simultaneous quantification of inflammatory markers in plasma samples without the need for nephelometric determination of these proteins.     

Carbohydrate-deficient transferrin (CDT) as a biochemical tool for the screening of congenital disorders of glycosylation (CDGs)

OBJECTIVE: The aim of this study was to provide a tool based on CDT measurements for a diagnostic approach to identify patients affected by congenital disorders of glycosylation (CDG) in a selected population. DESIGN AND METHODS: Serum carbohydrate-deficient transferrin (CDT) of pediatric and adult patients (a total of 168 individuals) with neurological symptoms was analyzed. Abnormal results were confirmed by HPLC analysis and by enzymatic and molecular studies. RESULTS: We found 6 patients (3.8%) with abnormal serum CDT; only two of them (1.9%) showed increased amounts of disialo and asialo with HPLC analysis and were classified as CDG-Ia, the most frequent form of CDG, due to a deficiency of the phosphomannomutase enzyme. CONCLUSIONS: The CDT quantitative immunoturbidimetric procedure is a useful tool for CDG screening. HPLC analysis, however, permitted the correct identification of asialo and disialo transferrin concentrations.     

全自动毛细管电泳仪在新生儿α珠蛋白生成障碍性贫血筛查中的应用

目的 探讨全自动毛细管电泳仪在新生儿α珠蛋白生成障碍性贫血筛查中的应用价值.方法使用全自动毛细管电泳仪对45 863例新生儿脐血标本进行血红蛋白(Hb)电泳分析,检测HbA、HbF、HbA2和异常Hb的含量,对筛查表型阳性的病例召回进行基因分析.结果 45 863例新生儿脐血标本中检测出巴特血红蛋白(Hb Bart′s)阳性者2 964例,筛查阳性率6.46%(2 964/45 863);召回其中1 278例行基因分析,1 173例确诊为α珠蛋白基因缺失,符合率91.8%.确诊病例以标准型为主,占64.4%;基因分型以--SEA/αα最为常见.静止型的Hb Bart′s含量95%置信区间为0.13%~0.97%,标准型Hb Bart′s含量95%置信区间为1.28%~5.92%,HbH病Hb Bart′s含量95%置信区间为8.32%~30.28%,Hb Bart′s水肿胎Hb Bart′s含量高于80%.结论 全自动毛细管电泳分析技术与基因分析有良好的一致性,根据Hb Bart′s含量的多少可初步进行α珠蛋白生成障碍性贫血的临床分型.     

毛细管电泳法和微芯片电泳法在糖类分析中的应用

糖类是三大生命物质之一,它参与多种生物代谢和其他生命活动.由于糖类的结构复杂,对它的研究方法要求较高.毛细管电泳以其快速、高效和高灵敏度、所需样品少等特点被广泛应用于糖类化合物的分析.而近几年快速发展的微芯片技术也在糖类分析中得到应用.本文就毛细管电泳法和微芯片电泳法在各类糖中的应用进行了阐述.     

V8 Nexus高速自动毛细管电泳仪血清蛋白电泳参考区间的建立

目的 初步建立V8 Nexus高速自动毛细管电泳仪(简称V8毛细管电泳仪)血清蛋白电泳(SPE)的参考区间.方法 选取表面健康者649名,按年龄分为18～29岁组、30～39岁组、40～49岁组、50～59岁组及≥60岁组.采用V8毛细管电泳仪[毛细管电泳(CE)法]检测所有对象的血清样本,依据我国卫生行业标准WS/T...     

利用毛细管电泳测定血清中维生素K的含量

目的 建立毛细管电泳定量检测血清中维生素K1和K2的方法。方法 利用固相萃取方法提纯血清中维生素K，将20mmol/L的四硼酸钠、10mmol/L的SDS加入到含有80％甲醇、10％去离子水、10％乙腈的溶液中配置成缓冲液，在254nm波长条件下对样本进行分离。结果 利用这种方法对血清中维生素K1和K2进行了有效的分离，该方法的线性低于病理状态下血清中维生素K的浓度，回收率为97％—103％，日内精密度(CVs)和日间精密度分别为7．16％—8．03％和10．08％—11．3％。结论 毛细管电泳方法测定血清中维生素K1和K2，具有快速、灵敏、定量检测等优点，可能成为测定人类血清中维生素K状态的有效检测方法。     

Fully automated continous flow system for determination of serum triglycerides (author's transl)

This report describes a fully automated continuous flow system for deter mining serum triglycerides in which a rapid, complete and specific hydrolysis o triglycerides to glycerol and fatty acids replaces the extraction and saponification procedures. After dialysis, the enzymatic determination of glycerol with glycerol-NAD oxidoreductase, is followed through absorbance changes at 340 nm.     

Conditions for single-strand conformation polymorphism (SSCP) analysis of BRCA1 gene using an automated electrophoresis unit.

The single-strand conformation polymorphism procedure has been applied in routine testing for hereditary diseases and cancer. However, temperature, running time, gel composition, fragment length, etc. can influence its sensitivity. Mutation detection in the clinical setting depends on the development of automated technology, especially for large genes such as the breast cancer gene BRCA1. We analysed DNA samples with BRCA1 mutations in an automated system (GenePhor System; Amersham-Pharmacia Biotech, Uppsala, Sweden). The concentrations of DNA template and PCR primers, the effect of chilling after denaturation, and the temperature and time of the electrophoresis were investigated. All band-shifts were detected by electrophoresis at 5 degrees C for 2 h 15 min. Concentrations of DNA and samples used in the PCR did not affect the SSCP pattern, but chilling the PCR product in ice after denaturation was required. The type and position of mutation in the fragments did not influence the probability of a mobility shift, although SSCP analysis was more sensitive for fragments shorter than 350 bp. This automated SSCP method meets the requirements of fast turnaround and sensitivity and can be readily adapted to the screening of large genes such as BRCA1.     

Carbohydrate-deficient transferrin (CDT) determination by nephelometry using a commercial kit. Analytical and diagnostic aspects.

Carbohydrate-deficient transferrin (CDT) has been proposed as the most efficient marker of alcohol abuse. Absolute and relative concentrations of CDT were measured with a commercial assay (%CDTTIA from AXIS-Shield, Oslo, Norway) using rate nephelometry for transferrin determination. One hundred eighty-eight alcoholic patients (154 males, 34 females) and 132 control patients (113 males, 19 females) were included in the study. Within-run and day-to-day imprecision were 3.15% and 9.77%, respectively. The calibration curve was stable for more than 4 months with a shift below 5%. The commercial assay lacked sensitivity (Se = 0.48), but was highly specific (Sp = 0.98). Lowering the cut-off from 6% to 4.6% raised the sensitivity of the %CDTTIA test to 0.76 with a specificity of 0.90. We conclude that this adaptation to the Array Protein System (Beckman-Coulter) is suitable for routine use and offers precise results. It, however, requires an adaptation of the cut-off value for patients and of the target value for kit controls.     

非浓缩尿SDS-琼脂糖凝胶蛋白电泳的临床应用

目的　探讨非浓缩尿蛋白电泳对早期肾损伤诊断的应用价值。方法　对 5 5例患者尿液标本分别进行尿蛋白半定量及尿蛋白 (U Pr)、尿微量白蛋白 (mAlb)、N 乙酰 β D 氨基葡萄糖苷酶 (NAG) ,β D 半乳糖苷酶(GAL)定量测定 ,与非浓缩尿的十二烷基磺酸钠 琼脂糖凝胶蛋白电泳 (SDS AGE)结果进行分析比较。结果　与SDS AGE结果相比较 ,尿常规及全自动生化仪定量测定有关指标的灵敏度相对较低。结论　SDS AGE检测灵敏度高 ,方法简便 ,省时 ,扫描后可得半定量结果 ,且结果较直观 ,对早期肾损伤诊断有较大的应用价值。     

Evaluation of a new automated system for the determination of ck-mb isoforms

We evaluated a new analyzer (Cardio REP) specifically designed for cardiac CK-MB isoenzyme and isoforms activity, with a performance time of 24 minutes. Ten AMI patients, with times elapsed between the onset of chest pain and admission to hospital ranging from 30 minutes to 4 hours, were monitored every 3–4 hours until the 16th hour of hospitalization. In each serum sample, in addition to total CK-MB and CK-MB isoforms measured by the Cardio REP analyzer, we also assayed total CK activity, CK-MB activity by immunoinhibition method, CK-MB mass concentration, CK-MB isoforms by REP method, troponin T, and myoglobin. The precision study demonstrated acceptable within assay and between assay CVs% for total CK-MB (8.1 and 10.4), MB₁ (9.1 and 14.2), and MB₂ (9.1 and 8.2) isoforms. The method was found to be linear up to 371 U/L for MB₂ isoform fraction and up to 516 U/L for total CK-MB. Results for CK-MB obtained with the Cardio REP correlated well with those for CK-MB activity obtained with the immunoinhibition method (r = 0.869) and those of CK-MB mass concentration (r = 0.923). The sensitivity of the Cardio REP CK isoforms method was found to be greater than that of the REP CK isoforms method. Time to first increased value of MB₂/MB₁ ratio and MB₂ isoform was earlier in comparison to that for CK-MB mass concentrations and similar to that for myoglobin, a marker that, however, lacks specificity. The diagnostic efficiency of CK-MB isoforms and the availability of a real-time, fully automated method for their measurement suggest the utilization of this biochemical marker in emergency for the early diagnosis of AMI.     

Preclinical evaluation of a semi‐automated and rapid commercial electrophoresis assay for von Willebrand factor multimers

Background

The von Willebrand factor (VWF) multimer test is required to correctly subtype qualitative type 2 von Willebrand disease (VWD). The current VWF multimer assays are difficult, nonstandardized, and time‐consuming. The purpose of this study was to evaluate the clinical utility of the commercial VWF multimer kit by Sebia (Lisses, France), an electrophoresis technique yielding same‐day results.

Methods

Ten healthy volunteer plasma samples, in‐house reference plasma (IRP) and commercial normal plasma (CNP) samples, 10 plasma samples from patients with a known VWD type, 1 hemophilia A plasma sample, and 7 external quality assurance (EQA) samples were analyzed using the commercial VWF multimer kit. Additional coagulation testing included measurements of VWF antigen (VWF:Ag), VWF activity (VWF:Ac), and FVIII activity (FVIII:C).

Results

The CNP results revealed a relative loss of the highest molecular weight multimers; therefore, IRP was preferred as the reference sample. The interpretations of 10 patients with a known VWD type could be successfully reproduced and agreed with previous VWF multimer results. In all EQA surveys, the multimer results and final VWD diagnosis agreed with expert opinion.

Conclusions

The VWF multimer assay by Sebia is easy to perform and can be successfully implemented in any clinical laboratory for second‐stage evaluation of VWD. The resolution power of multimer distribution is adequate to correctly classify VWD types 1, 2A, 2B, and 3.

     

Quantification of urinary allantoin by capillary zone electrophoresis during recombinant urate oxydase (rasburicase) therapy

OBJECTIVES: Rasburicase (Fasturtec) is used to prevent or treat hyperuricemia associated with chemotherapy. We developed a capillary zone electrophoresis method to measure urinary allantoin, the degradation product of uric acid by rasburicase. DESIGN AND METHODS: Electrophoresis was performed using a P/ACE 5500 system (Beckman) with a fused silica capillary tube and a UV-visible detector set at 214 nm. Urine samples from 10 patients with non-Hodgkin's lymphoma were analyzed to validate the technique. RESULTS: Using a sodium tetraborate running buffer, urinary allantoin was separated from related compounds and internal standard in less than 30 min. The method was linear up to 1.25 g/L (quantification limit: 30 mg/L); precision was below 10%. The total amount of allantoin excreted in patients treated by rasburicase ranged from 1.5 g to 7.9 g/4 days. CONCLUSION: This CZE assay is a simple, rapid and reproducible method to measure allantoin in urine. Different elimination profiles have been found in patients treated with rasburicase.     

应用高效毛细管电泳筛检和鉴定血清中M蛋白

目的:应用毛细管区带电泳法(CZE)和毛细管区带电泳-免疫削减法(IS-CZE)对M蛋白进行筛检和鉴定。方法:运用CZE和琼脂糖凝胶电泳(AGE)分别对532例血清进行M蛋白的筛检;用IS-CZE和免疫固定电泳(IFE)对M蛋白阳性的64例标本进行免疫分型。结果:与AGE相比较,CZE筛检M蛋白的阳性率为96.9%;与IFE相比较,IS-CZE分型的符合率为89.1%;自包被固相抗体能满足试验要求。结论:毛细管区带电泳可高效、快速完成M蛋白的筛检和鉴定,适合临床推广。     

应用脉冲场凝胶电泳分型技术对江西省伤寒菌株的分析

目的 分析江西省1986年以来伤寒爆发疫情菌株的分子分型特征.方法 对所收集的伤寒沙门菌株利用脉冲场凝胶电泳(PFGE)技术进行菌株的分子分型.结果 40株伤寒菌株分离自1986、1987、2000和2003年的伤寒患者和带菌者,利用PFGE可分为16个型.不同年份、不同地点爆发的伤寒菌株带型不同,同一年份同一地点爆发的伤寒菌株带型也有差异.结论 基于PFGE的分子分型能很好地应用于菌株特征的分析,伤寒杆菌在不同年份变异较大.常规的疫苗是否能应对不同PFGE型菌株的流行值得关注.     

Evaluation of a new capillary zone electrophoresis system for the identification and typing of Bence Jones Protein

OBJECTIVES: Capillary electrophoresis has recently emerged as a new sensitive technique for the separation of urinary proteins. We evaluated a new method for Bence Jones Protein (BJP) detection and characterization on native urine samples by the Paragon CZE 2000 system. To avoid interference in electrophoretic separation, urine samples were preliminarily treated for the selective removal of interfering salt particles. DESIGN AND METHODS: The evaluation was done on a total of 350 fresh 24-h urine samples. The salt particle removal consisted of a manual chromatographic separation, optimized in the course of our evaluation. Capillary zone urinary protein electrophoresis (CZ-UPE) was compared with conventional high-resolution electrophoresis on an agarose gel, while capillary immunosubtraction (CZU-IFE) was compared with agarose gel immunofixation. RESULTS: After finding a consistent protein loss in eluates, the preanalytical treatment was optimized by changing sample dilution and eluate collection. The within- and between-run imprecision values for monoclonal peaks corresponding to BJP ranged from 0.4-12.2% to 3.3-6.3%, respectively. The detection limit for BJP, defined as the lowest measurable monoclonal peak on CZ-UPE, was 0.0012 g/L for kappa BJP and 0.0007 g/L for lambda BJP. CZ-UPE and CZU-IFE sensitivities were significantly lower in urine samples with a total protein level < or = 100 mg/L (67% and 78%, respectively) compared to those with total protein >100 mg/L (92% and 94%, respectively). Comparison between BJP measurements obtained from densitometric scanning with those from absorbance tracing showed a correlation coefficient of 0.994 and a bias of 29.8 mg/L. CONCLUSIONS: Paragon CZE 2000 can be introduced in routine for screening and typing of BJP; in urine samples with a total protein level >100 mg/L, the performance is consistent with results from published validation studies on CZE applied to serum samples.