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Capillary isoelectric focusing of hemoglobin variants in the pediatric clinical laboratory
Author(s) -
Hempe James M.,
Granger Jerry N.,
Craver Randall D.
Publication year - 1997
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150181013
Subject(s) - isoelectric focusing , chromatography , hemoglobin , capillary electrophoresis , chemistry , hemoglobin variants , capillary action , whole blood , dried blood spot , biochemistry , medicine , immunology , materials science , composite material , enzyme
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) post‐translationally modified Hb present at low concentrations in normal blood, (ii) Hb oxidation products produced by improper sample storage, (iii) differential diagnosis of S/β + thalassemia, G‐Philadelphia trait, S/C‐Harlem disease, and Hb H disease, (iv) sensitive detection of minor variants like Hb A 2 ′ as indicators of an α 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.