TY - JOUR
T1 - New ultra-micro high-performance liquid chromatographic method for determining the γ chain composition of hemoglobin F in normal adults
AU - Kutlar, F.
AU - Huisman, T. H.J.
PY - 1993/10/29
Y1 - 1993/10/29
N2 - 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.
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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U2 - 10.1016/0378-4347(93)80002-L
DO - 10.1016/0378-4347(93)80002-L
M3 - Article
C2 - 7507934
AN - SCOPUS:0027486127
SN - 0378-4347
VL - 620
SP - 183
EP - 189
JO - Journal of Chromatography B: Biomedical Sciences and Applications
JF - Journal of Chromatography B: Biomedical Sciences and Applications
IS - 2
ER -