Structural characterization of abnormal hemoglobins from dried blood specimens in a neonatal screening program

Abstract

Blood specimens dried on filter paper are now widely used for neonatal screening of hemoglobinopathies. These samples are perfectly suited for electrophoresis studies and HPLC analysis. They may also be used for DNA analysis. The structural characterization of a hemoglobin variant is also possible using protein chemistry methods. After elution of the hemoglobin from the paper, the different components are fractionated by a microscale preparative isoelectric focusing. The structural modification of the abnormal hemoglobin is then determined through a series of techniques including chain separation, aminoethylation, trypsin digestion, analysis of the peptides and determination of their aminoacid composition. The efficiency of this strategy is demonstrated by the study of an alpha-chain variant (Hb Hasharon) and three beta-chain variants (Hb S, Hb D Punjab, Hb E). Unambiguous identification of the structural abnormality was obtained with samples stored for up to 18 months and with abnormal fractions amounting to only approximately 10% of the total lysate.