Interference of hemoglobin Hope on beta-thalassemia diagnosis by the capillary electrophoresis Method

The β-chain hemoglobin (Hb) variants interfere with the diagnosis of β-thalassemia trait using high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE). We analyzed the effect of Hb Hope, a β-chain Hb variant frequently found in the Thai population, on β-thalassemia trait diagnosis. HPLC and CE were used to quantify the level of HbA(2) in 11 whole blood samples containing Hb Hope. The levels of Hb Hope detected by both methods were similar. An elevated HbA(2) level was found in all samples analyzed by the CE method, while 1 was increased when analyzed by HPLC, which was a compound heterozygous of Hb Hope and α-thalassemia-1 SEA-type deletion. Of 11 samples, 6 had mean corpuscular volumes within the reference range. All samples showed negative results for molecular analysis of β(0)-thalassemia codon 17, 41/42, and 71/72 mutations and β-thalassemia 3.5-kb deletion. Therefore, Hb Hope interfered with the diagnosis of β-thalassemia trait analyzed by CE but not by HPLC.     

毛细管等电聚焦电泳定量分析健康成人及地中海贫血携带者的Hb A2

目的 对等电聚焦毛细管电泳(capillary isoelectric focusing，CIEF)检测血红蛋白A2(HbAt)含量做出用于筛查地中海贫血(地贫)携带者的方法学评价。方法 收集105名健康成人、93例经常规地贫筛查有阳性表型的连续临床样品。通过正常人样品分析比较CIEF测定HbA2与Helena Spife combo电泳系统结果的一致性；通过比较样品内和样品间Hb A2的CIEF测定值的变异系数评价该法的精确度和稳定性；通过分析阳性样品的基因型来评价CIEF检测地贫的准确性。结果 用CIEF分析当地健康人的Hb A2正常参考值为3．59％～5．23％。CIEF结果与常规血红蛋白电泳结果具有良好的线性关系；以基因分析为确诊标准，用CIEF检测Hb A2含量，及结合红细胞指数阳性测定值所筛查的87例α-地贫或β-地贫携带者、6例成人血红蛋白E病例样品均被准确诊断，未见假阳性或假阴性结果。结论 等电聚焦毛细管电泳是一种快速高效、高稳定性、高精确度的HbA2测定方法，可用作地贫携带者的常规筛查手段。     

Reliable estimation of hemoglobin A2 concentration by electrophoresis with densitometry.

Elevated hemoglobin A2 (Hb A2) levels can be identified conveniently by densitometry after electrophoresis on cellulose acetate strips. Because a recent report questioned the accuracy of this technic, the method was re-evaluated by paired comparison with microcolumn chromatography. Analysis of 100 patient specimens showed high correlation (r = +0.84), but an average Hb A2 concentration 0.7% higher by densitometry than by chromatography (P less than 0.001). With upper limits set at 4.5%, and 3.8%, respectively, results were divided into "normal" and "high" for each method. Concordant results were obtained in 97 of the 100 cases (82 normal, 15 high). Another densitometer of improved design was used for paired analysis of 50 additional specimens, 25 normal and 25 with beta-thalassemia trait. The two groups were well separated by both procedures, and Hb A2 levels were similar (r = +0.92, P greater than 0.6). This study demonstrates that it is possible, with carefully controlled technics and properly calibrated instruments, to use electrophoresis with densitometry as a reliable means of identifying abnormal Hb A2 levels.     

Characterization of poly(ethylene glycol)-modified bovine hemoglobin by capillary zone electrophoresis

In this study capillary zone electrophoresis (CZE) was used to analyze the poly(ethylene glycol)-modified bovine hemoglobin(PEG-bHb). The results show that CZE separated the subunit of bovine hemoglobin based on the number of PEG conjugating to the protein surface, which makes it possible to evaluate the degree of modification of hemoglobin subunit; meanwhile, it also reflected the stability of PEG attaching to hemoglobin after incubating with hydroxylamine, which makes it successful to detect the distribution of attachment site and evaluate the stability of PEG on the surface of hemoglobin. As a simple, fast and accurate method, CZE is suitable to monitor the production procedures and quality control of the final products of the PEG-bHb.     

4411例新生儿血红蛋白毛细管电泳地中海贫血筛查的探讨

目的探讨新生儿血红蛋白毛细管电泳地中海贫血筛查的应用价值。方法对采用毛细管血红蛋白电泳筛查新生儿地中海贫血资料进行回顾性总结。结果4411例新生儿血红蛋白毛细管电泳筛查,检出异常血红蛋白523例,阳性率11．89％,其中,检出d地中海贫血473例（其中HbBart’s胎儿水肿综合症1例,出生时已死亡：HbBart’s含量〈70％为467例;HbCS5例）,阳性率10．72％。β-地中海贫血15例（HbE杂合子15例）,阳性率0．34％。检出异常血红蛋白病（即HbD和HbJ杂合子）35例,阳性率0．79％。结论新生儿脐血红蛋白毛细管电泳地中海贫血筛查主要以d为地中海贫血主,β-地中海贫血很难筛查,除非出现HbE带,筛查阳性者建议做地贫基因检测或DNA序列测序分析。     

Interference with hemoglobin A(1C) determination by the hemoglobin variant Shelby

Hemoglobin variant carrier status was found in a 46-year-old African American man following detection of a falsely elevated hemoglobin A1c (HbA1c) by ionexchange high-performance liquid chromatography (HPLC, VARIANT A1c, Bio-Rad Laboratories, Hercules, CA). Additional analysis of the hemoglobin variant using the Beta Thal Short program (Bio-Rad) revealed an unknown peak with a retention time of 4.84 minutes and a proportion of 26.3%. No mass shift in alpha-globin or beta-globin proteins was observed by mass spectrometry. DNA sequencing revealed a missense mutation in 1 beta-globin allele corresponding to the hemoglobin Shelby trait. The patient was asymptomatic with a normal hemoglobin value of 13.6 g/dL (136 g/L) but had increased target cells on a peripheral blood smear. An alternative method for HbA1c determination using boronate-affinity HPLC provided a value of 3.9% (0.04; reference range, 4.0%-6.9% [0.04-0.07]), more consistent with the patient's recent blood glucose values in the normal range.     

芯片电泳相对迁移时间DNA长度计算方法鉴定高危型人乳头状瘤病毒基因

目的 建立一种能够消除电泳分析中迁移时间漂移对于DNA片段长度预测精确性影响的芯片电泳相对迁移时间比例方法,并联用多重PCR鉴定高危型人乳头状瘤病毒(HPV).方法 在芯片电泳分析中引入上位和下位内标,依据待测DNA片段相对于上位及下位内标的迁移时间比例进行长度预测,建立芯片电泳相对迁移时间比例的测定方法.考察不同实验条件下芯片电泳DNA分析中相对迁移时间比例的重现性和DNA片段长度预测精确性.选取2007年10月至2008年12月间广州医学院附属广州市第一人民医院妇科就诊的114例患者的富颈上皮细胞样品,经细胞学检查划分为正常组(n=30)、不典型鳞状细胞组(ASCUS组,n=30)、低度鳞状上皮内病变组(LSIL组,n=30)和高度鳞状上皮内病变和(或)鳞状细胞癌组[HSIL和(或)SCC组,n=24].对应活检组织经病理诊断,划分为宫颈高度病变组(n=90)和对照组(n=24).利用多重PCR联合芯片电泳相对迁移时间比例方法检测宫颈上皮细胞样本中5种高危型HPV感染状况.参考组织病理学检测结果评价芯片电泳相对迁移时间比例的方法分析对于宫颈高度病变诊断的临床价值.结果 芯片电泳相对迁移时间比例方法具有良好的重现性和DNA片段长度相关性.使用芯片电泳相对迁移时间比例方法能够满足HPV基因分型多重PCR产物的准确鉴定.各个细胞学分类级别对应的5种高危HPV检出率分别为正常组33.3％、ASCUS组60％、LSIL组76.6％、HSIL和(或)SCC组83.3％.HPV感染率随细胞和组织学病变级别增加有上升趋势,与细胞学(x2=19.319,P＜0.05)和组织学(x2=7.268,P＜0.05)分级具有明显的关联.结论 芯片电泳相对迁移时间比例方法简单可靠,可以实现高危HPV基因型快速判定,具有筛查宫颈高度病变的价值.     

Quantitation of A2 hemoglobin by polyacrylamide gel disc electrophoresis: a method with individual specimen standardization

A polyacrylamide gel electrophoresis technic for the determination of hemoglobin A2 is presented. Rapid separation is an advantage. The use of a diluted hemolysate as a 4 percent standard avoids overestimation of the A2 fraction by densitometry and also provides a reference for visual comparison. Normal range of A2 hemoglobin by this method is 1.12 to 4.13 per dkg. Coefficient of variation was 5.84 percent.     

Analysis of small-scale biological compartments by capillary electrophoresis

Two characteristics of capillary electrophoresis make the technique attractive for the separation of the components of microscale compartments within living organisms: small sample volume requirements and direct compatibility with biofluids. Indeed, capillary electrophoresis has been used for analysis down to a sub-cellular level. There are also potentially many applications of capillary electrophoresis to biological compartments on a super-cellular scale, which are nevertheless so small that they make analysis by conventional separations techniques difficult or impractical. The analytical challenges in small-scale bioanalysis are first to develop a suitable method for collection of sample and its introduction into the separation capillary, and secondly, to achieve the required separation. Examples reviewed here will primarily focus on the analysis of tear fluid or airway surface liquid, cases in which the amount of sample that can be collected range from around 10 microl to around 100 nl.     

Analysis of cyanogenic glycosides by micellar capillary electrophoresis

The separation of amygdalin, prunasin and their isomers neoamygdalin and sambunigrin could be achieved with micellar capillary electrophoresis (MEKC). The two isomers were obtained in alkaline conditions and were produced in less than 15 min at pH 11.0. The developed methods showed a good selectivity in the separation of the isomers only in the presence of SDS micelles. The working pH was optimized to allow best resolution and quantitative analysis of these compounds. With a linear calibration over an injection time from 1 to 20 s, the detection limit was found to be in the range of 5 microM (S/N=3; 20 s injection time). Two pH buffer systems (pH 5.2 and pH 9.1) were chosen to confirm the peak attributions of the compounds in the apple and peach seeds samples. Sambunigrin was found in both apple and peach seeds but could not be quantified because of missing standards. Prunasin and amygdalin were not found in the apple sample, while they were quantified in the peach seeds in concentrations of 50 microg/g and 90 microg/g (dry weight), respectively.     

Measurement of nephrolithiasis urinary markers by capillary electrophoresis

A previously developed method for screening organic acidurias by capillary electrophoresis has been validated for oxalate and citrate measurement in urine. Sample pretreatment is minimum, just acidification and centrifugation. Detection is by direct UV. Validation parameters of the method can be considered adequate. Response is linear for both analytes in standards and samples. The assayed ranges were 200-1,000 mg/l for citrate and 10-200 mg/l for oxalate. Recoveries ranged from 99.4+/-3 to 101.7+/-2.4%, maximum imprecision in oxalate concentration was of 7.6% RSD and limits of detection in samples were 0.67 mg/l for oxalate and 25.9 mg/l for citrate, both lower than the measured values in samples. Identification of increased glyoxylic (oxoacetic acid) and glyceric acids (2,3-dihydroxy propanoic) are also included to facilitate the diagnosis.     

Determination of pyrroloquinoline quinone by capillary zone electrophoresis

A new method for the determination of pyrroloquinoline quinone by capillary zone electrophoresis has been developed. Separation conditions have been optimised with the respect to different parameters including pH and ionic strength of the background electrolyte, separation voltage and temperature of the capillary. A buffer consisting of 50 mM beta-alanine-HCl pH 3.0 was found to be the most suitable electrolyte for this separation. An applied voltage of 25 kV (negative polarity) and a temperature of 25 degrees C gave the best analysis of pyrroloquinoline quinone. The linear detection range for concentration versus peak area for the assay is from 5 to 500 microM (correlation coefficient 0.9998) with a detection limit of 0.1-0.2 microM. The inter-day reproducibility of the peak area was 2.5% and the inter-day reproducibility of the migration time was below 0.18%.     

Fast analysis of antibacterial isothiazolones by capillary electrophoresis

Some technical aspects influencing the total time of CE analysis are discussed. A high throughput electrophoretic system based on micellar electrokinetic chromatography (MEKC) is demonstrated as an example. A short capillary, strong electric field, alkaline buffer (pH 9.5) generating strong electroosmotic flow, and parallel hydrodynamic pressure allow the separation of two uncharged isothiazolone derivatives within 45 s.     

Comparison of microchromatography and electrophoresis with elution for hemoglobin A2 (Hb A2) quantitation.

Microcolumns prepared in the authors' laboratory, two commercial microchromatography kits, and electrophoresis with elution were compared for Hb A2 quantitation. Day-to-day imprecision of microchromatographic methods was similar (CV 4.7--6.6%) and somewhat less than electrophoresis with elution (CV 8.0--9.1%). Both commercial kits showed variable imprecision in different lots; one lot of Kit B gave erratic results due to resin leakage. From 49 patient specimens, Kit A microcolumns and those of the authors identified the same 14 patients with an elevated percentage of Hb A2 and showed good correlation (P = 0.90), although Kit A showed constant bias toward higher values. Electrophoresis with elution resulted in a false-positive and a false-negative value, did not correlate well with microcolumns (P = 0.78 and 0.76), and showed proportional bias toward lower values for an elevated percentage of Hb A2. Commercial kits were convenient, relatively quick, and cost-effective. Frozen, stabilized hemolysates performed well for quality control.     

高效毛细管电泳法分析rhIL—2的纯度

重组人白细胞介素 2 ( rh IL - 2 )是利用基因工程技术 ,从大肠杆菌中获得的。 rh IL- 2能抑制肿瘤细胞的增殖 ,是肿瘤过继免疫治疗中应用最广泛 ,研究得最多的细胞因子 [1 ] 。作为生物制剂 ,在药品的质量监控中 ,其纯度分析又是非常重要的指标之一。目前 ,常用的纯度鉴定方法有凝胶电泳和高效液相色谱 ( HPL C)。与 HPL C相比 ,高效毛细管电泳( HPCE)具有更高的分辨率和灵敏度 ,且同时具有快效、高效、自动化、上样量少等特点 [2 ] 。本文运用 HPCE对 rh IL - 2进行纯度分析 ,建立了快速简便的测定方法。1　材料与方法1.1　仪器…     

Chiral analysis of methylphenidate and dextromoramide by capillary electrophoresis

A HPLC method is described to quantify picolinic acid in milk, blood serum and tissue culture supernatant. The method requires very little sample preparation because acid precipitation allows total recovery of picolinic acid. High specificity and sensitivity were obtained using ion-pair chromatography on a C18 reversed-phase column with tetrabutylammonium hydrogen sulfate as ion pairing reagent. We describe the conditions for the automated testing of multiple samples and for the detection of L-tryptophan and L-kynurenine together with picolinic acid. This system will be utilized to elucidate the relationship between picolinic acid production and human disease. Furthermore, we provide the first evidence of picolinic acid in human blood serum.