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HPLC determination of hemoglobins
to establish reference values with the aid
of statistics and informatics
L.S. Ondei1, P.J.A. Zamaro1, P.H. Mangonaro2, C.R. Valêncio2 and C.R. Bonini-Domingos1
1Laboratório de Hemoglobinas e Genética das Doenças Hematológicas, Departamento de Biologia, IBILCE/UNESP, São José do Rio Preto, SP, Brasil
2Grupo de Banco de Dados, Departamento de Ciências da Computação e Estatística, IBILCE/UNESP, São José do Rio Preto, SP, Brasil
Corresponding author: L.S. Ondei
E-mail: luondei@yahoo.com.br
Genet. Mol. Res. 6 (2): 453-460 (2007)
Received January 19, 2007
Accepted May 28, 2007
Published June 30, 2007
ABSTRACT. The purpose of the present study was to establish reference values for hemoglobins (Hb) using HPLC, in samples containing normal Hb (AA), sickle cell trait without alpha-thalassemia (AS), sickle cell trait with alpha-thalassemia (ASH), sickle cell anemia (SS), and Hb SC disease (SC). The blood samples were analyzed by electrophoresis, HPLC and molecular procedures. The Hb A2 mean was 4.30 ± 0.44% in AS, 4.18 ± 0.42% in ASH, 3.90 ± 1.14% in SS, and 4.39 ± 0.35% in SC. They were similar, but above the normal range. Between the AS and ASH groups, only the amount of Hb S was higher in the AS group. The Hb S mean in the AS group was 38.54 ± 3.01% and in the ASH it was 36.54 ± 3.76%. In the qualitative analysis, using FastMap, distinct groups were seen: AA and SS located at opposite extremes, AS and ASH with overlapping values and intermediate distribution, SC between heterozygotes and the SS group. Hb S was confirmed by allele-specific polymerase chain reaction. The Hb values established will be available for use as a reference for the Brazilian population, drawing attention to the increased levels of Hb A2, which should be considered with caution to prevent incorrect diagnoses.
Key words: Reference values, High-performance liquid chromatography, Hemoglobin
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