Hempe J M, Granger J N, Craver R D
Department of Pediatrics, Louisiana State University School of Medicine, New Orleans 70112, USA.
Electrophoresis. 1997 Sep;18(10):1785-95. doi: 10.1002/elps.1150181013.
We have used capillary isoelectric focusing (cIEF) to diagnose and monitor hemoglobinopathies in children for over three years. This report describes a major revision of our original method (Clin. Chem. 1994, 40, 2288-2295) that improves the analysis of hemoglobin (Hb) variants by cIEF, especially capillary performance and quantitation precision for minor variants. The revised method uses mixed ampholytes (2% pH 6-8:3-10; 10:1), lower viscosity methylcellulose (0.375%) solution, between-sample capillary conditioning with methanol, and hemolysates prepared from red blood cells (RBC) instead of whole blood. Collectively, these changes prolonged capillary life by minimizing capillary exposure to NaOH, standardized the sample matrix, and improved the precision of peak autointegration. The between-run quantitation imprecision (% relative standard deviation) of the revised method was 0.1-3.5% for all diagnostically important major and minor Hb variants present at normal or abnormal levels. The results show the use of the revised method for (i) posttranslationally modified Hb present at low concentrations in normal blood, (ii) Hb oxidation products produced by improper sample storage, (iii) differential diagnosis of S/beta + thalassemia, G-Philadelphia trait, S/C-Harlem disease, and Hb H disease, (iv) sensitive detection of minor variants like Hb A2' as indicators of an alpha globin mutation, and (v) neonatal screening using dried blood collected on filter paper. The results show that high-efficiency separation and precise quantitation of Hb variants over a wide range of concentrations makes cIEF a comprehensive assay that can be used without adjunct analyses for the automated primary evaluation of hemoglobinopathies and thalassemias.
三年多来，我们一直使用毛细管等电聚焦（cIEF）技术来诊断和监测儿童血红蛋白病。本报告描述了对我们原方法（Clin. Chem. 1994, 40, 2288 - 2295）的重大修订，该修订改进了通过cIEF对血红蛋白（Hb）变体的分析，特别是对微量变体的毛细管性能和定量精度。修订后的方法使用混合两性电解质（2% pH 6 - 8:3 - 10；10:1）、较低粘度的甲基纤维素（0.375%）溶液、用甲醇进行样品间毛细管预处理，以及用红细胞（RBC）而非全血制备的溶血产物。总体而言，这些改变通过尽量减少毛细管与NaOH的接触延长了毛细管寿命，使样品基质标准化，并提高了峰自动积分的精度。对于所有正常或异常水平的具有诊断重要性的主要和次要Hb变体，修订后方法的批间定量不精密度（相对标准偏差%）为0.1 - 3.5%。结果表明，修订后的方法可用于：（i）正常血液中低浓度存在的翻译后修饰Hb；（ii）因样品储存不当产生的Hb氧化产物；（iii）S/β +地中海贫血、G -费城特征、S/C -哈勒姆病和Hb H病的鉴别诊断；（iv）灵敏检测如Hb A2'等微量变体作为α珠蛋白突变的指标；（v）使用滤纸上采集的干血进行新生儿筛查。结果表明，在广泛的浓度范围内对Hb变体进行高效分离和精确定量，使得cIEF成为一种全面的检测方法，无需辅助分析即可用于血红蛋白病和地中海贫血的自动化初步评估。
