New ultra-micro high-performance liquid chromatographic method for determining the γ chain composition of hemoglobin F in normal adults

F. Kutlar, T. H J Huisman

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10 Citations (Scopus)

Abstract

The difficulty in isolating the minute quantity of Hb F (<1%) present in the red blood cells of normal adults greatly complicates the determination of its γ chain composition. We have developed a rapid ultra-micro high-performance liquid chromatographic (HPLC) method and used it to analyze the γ chain composition of Hb F in 47 adults with Hb F levels between 0.1-3.4%. The method involves the isolation of Hb F from as little as 50 μl of whole blood on an analytical size cation-exchange HPLC column, followed by concentration in a Centricon micro concentrator unit and by reversed-phase HPLC analysis. The entire procedure can be completed in one day and 3-4 analyses can be made simultaneously. We reanalyzed the blood samples from 22 subjects with known β-globin gene cluster haplotypes, and confirmed the association of high, low, and very low Gγ levels with haplotypes A, B, and C, respectively. Also included are the results of DNA sequence analyses of the Gγ and β promoters, and of the locus-control-region hypersensitive site-2 (LCR-HS-2) of the β-globin gene cluster in five subjects homozygous for haplotypes A, B or C; the data obtained failed to provide a satisfactory explanation for all the variations in the Gγ levels that have been observed.

Original languageEnglish (US)
Pages (from-to)183-189
Number of pages7
JournalJournal of Chromatography B: Biomedical Sciences and Applications
Volume620
Issue number2
DOIs
StatePublished - Oct 29 1993

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Fetal Hemoglobin
Haplotypes
Blood
Globins
Multigene Family
Liquids
Genes
Chemical analysis
Locus Control Region
DNA sequences
High performance liquid chromatography
Reverse-Phase Chromatography
DNA Sequence Analysis
Cations
Erythrocytes
High Pressure Liquid Chromatography
Cells
Association reactions

ASJC Scopus subject areas

  • Chemistry(all)
  • Clinical Biochemistry
  • Molecular Medicine

Cite this

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abstract = "The difficulty in isolating the minute quantity of Hb F (<1{\%}) present in the red blood cells of normal adults greatly complicates the determination of its γ chain composition. We have developed a rapid ultra-micro high-performance liquid chromatographic (HPLC) method and used it to analyze the γ chain composition of Hb F in 47 adults with Hb F levels between 0.1-3.4{\%}. The method involves the isolation of Hb F from as little as 50 μl of whole blood on an analytical size cation-exchange HPLC column, followed by concentration in a Centricon micro concentrator unit and by reversed-phase HPLC analysis. The entire procedure can be completed in one day and 3-4 analyses can be made simultaneously. We reanalyzed the blood samples from 22 subjects with known β-globin gene cluster haplotypes, and confirmed the association of high, low, and very low Gγ levels with haplotypes A, B, and C, respectively. Also included are the results of DNA sequence analyses of the Gγ and β promoters, and of the locus-control-region hypersensitive site-2 (LCR-HS-2) of the β-globin gene cluster in five subjects homozygous for haplotypes A, B or C; the data obtained failed to provide a satisfactory explanation for all the variations in the Gγ levels that have been observed.",
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AB - The difficulty in isolating the minute quantity of Hb F (<1%) present in the red blood cells of normal adults greatly complicates the determination of its γ chain composition. We have developed a rapid ultra-micro high-performance liquid chromatographic (HPLC) method and used it to analyze the γ chain composition of Hb F in 47 adults with Hb F levels between 0.1-3.4%. The method involves the isolation of Hb F from as little as 50 μl of whole blood on an analytical size cation-exchange HPLC column, followed by concentration in a Centricon micro concentrator unit and by reversed-phase HPLC analysis. The entire procedure can be completed in one day and 3-4 analyses can be made simultaneously. We reanalyzed the blood samples from 22 subjects with known β-globin gene cluster haplotypes, and confirmed the association of high, low, and very low Gγ levels with haplotypes A, B, and C, respectively. Also included are the results of DNA sequence analyses of the Gγ and β promoters, and of the locus-control-region hypersensitive site-2 (LCR-HS-2) of the β-globin gene cluster in five subjects homozygous for haplotypes A, B or C; the data obtained failed to provide a satisfactory explanation for all the variations in the Gγ levels that have been observed.

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