Application of cellulose acetate electrophoresis to globin chain separation for antenatal diagnosis of beta thalassemia

Boccacci, M.; Massa, A.; Tentori, L

doi:10.1016/0009-8981(81)90016-4

Cited by 11 publications

(4 citation statements)

References 6 publications

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“… 24 Moreover, the relative purity of globin chains in blood and erythroid samples renders antibody recognition unnecessary for the specific purpose of globin-chain detection, as hemoglobin makes up 99% of erythroid dry mass. 25 This knowledge is exploited by routine diagnostic methods widely applied for population screening of hemoglobinopathies, such as cation-exchange high-performance liquid chromatography (HPLC), 26 isoelectric focusing, 27 or cellulose acetate electrophoresis, 28 which rely on the detection of hemoglobin tetramers and, particularly if applied in combination, 29 allow sensitive detection of different hemoglobin variants. However, those methods give limited information on the specific quantities of constituting chains, in particular for the γ-globin chains, A γ and G γ, which differ by a single amino acid.…”

Section: Introductionmentioning

confidence: 99%

Rapid and Sensitive Assessment of Globin Chains for Gene and Cell Therapy of Hemoglobinopathies

Loucari

Patsali

Dijk

et al. 2018

Human Gene Therapy Methods

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The β-hemoglobinopathies sickle cell anemia and β-thalassemia are the focus of many gene-therapy studies. A key disease parameter is the abundance of globin chains because it indicates the level of anemia, likely toxicity of excess or aberrant globins, and therapeutic potential of induced or exogenous β-like globins. Reversed-phase high-performance liquid chromatography (HPLC) allows versatile and inexpensive globin quantification, but commonly applied protocols suffer from long run times, high sample requirements, or inability to separate murine from human β-globin chains. The latter point is problematic for in vivo studies with gene-addition vectors in murine disease models and mouse/human chimeras. This study demonstrates HPLC-based measurements of globin expression (1) after differentiation of the commonly applied human umbilical cord blood–derived erythroid progenitor-2 cell line, (2) in erythroid progeny of CD34+ cells for the analysis of clustered regularly interspaced short palindromic repeats/Cas9-mediated disruption of the globin regulator BCL11A, and (3) of transgenic mice holding the human β-globin locus. At run times of 8 min for separation of murine and human β-globin chains as well as of human γ-globin chains, and with routine measurement of globin-chain ratios for 12 nL of blood (tested for down to 0.75 nL) or of 300,000 in vitro differentiated cells, the methods presented here and any variant-specific adaptations thereof will greatly facilitate evaluation of novel therapy applications for β-hemoglobinopathies.

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Section: Introductionmentioning

confidence: 99%

Rapid and Sensitive Assessment of Globin Chains for Gene and Cell Therapy of Hemoglobinopathies

Loucari

Patsali

Dijk

et al. 2018

Human Gene Therapy Methods

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“…Traditionally, cellulose acetate electrophoresis after sample extraction and denaturation [6] is used for the separation of the globin chains. Several electrophoretic methods based on CE in acidic [7][8][9][10] or basic buffers [11] have been described for the analysis of the globin chains including those based on capillary isoelectric focusing [12].…”

Section: Introductionmentioning

confidence: 99%

Simplified hemoglobin chain detection by capillary electrophoresis

Shihabi¹,

Hinsdale²

2005

Electrophoresis

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Hemoglobin (Hb) chains have been analyzed traditionally by cellulose acetate electrophoresis after sample extraction with acetone and denaturation with concentrated urea in order to detect thalassemia (Thal). A few capillary electrophoresis (CE) methods have been also described for separation of Hb chains also after sample extraction. We describe a CE method for analysis of Hb chains without sample preparation. Red blood cells were diluted (hemolyzed) in water and injected directly onto the capillary. The separation was performed in concentrated phosphate buffer at pH 12.6 and 2.15. Under these conditions of pH and buffer concentration, the chains were denatured and separated from the heme during electrophoresis. The common variants of the beta-chains, such as beta(S), beta(C), and beta(E), are also separated from each other. The intact Hb molecule is analyzed using the same sample and CE conditions but in an arginine-Tris buffer, pH 8.6. The data from the three separations are used to complement each other for interpretation of the presence of Hb variants and for thalassemia. The main advantages of this method are simplicity and speed. This method illustrates the flexibility and simplicity of the CE for analysis of the hemoglobinopathies.

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“…The fetal reticulocytes are labeled in vitro and the radioactive globin chains analyzed by different chromatographic or electrophoretic techniques. The widespread carboxymethyl cellulose (CMC) chromatographic procedure of Clegg et a1 [2] has generally been replaced by faster methods, such as highpressure liquid chromatography (HPLC) [3], isoelectric focusing (IEF) [4,5], and electrophoresis on polyacrylamide gels [6] or on cellulose acetate membranes [7,8].…”

Section: Introductionmentioning

confidence: 99%

Antenatal diagnosis of β‐thalassemia by isoelectric focusing in immobilized pH gradients

Manca¹,

Cossu²,

Angioni

et al. 1986

American J Hematol

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A new method for antenatal diagnosis of thalassemias is reported based on the analysis of the major Hb components of fetal cord blood, sampled at week 18 of pregnancy under ultrasonic guidance, by isoelectric focusing in immobilized pH gradients (IPG). In an IPG gel encompassing a pH 6.7-7.6 span, HbA and HbFac are separated by a distance nine times greater than in a conventional carrier ampholyte pH 6-8 gel and three times greater than in an ampholine gel with separators (an equimolar mixture of beta-alanine and 6-amino caproic acid). Band evenness (in terms of uniform protein concentration within a zone) and straightness (in terms of parallel alignment of the bands to the electrodes), because of insensitivity of IPG gels to salt distortions, allows for accurate and reproducible quantitation of HbF, -A, and -Fac levels. The possibility of greatly overloading IPG matrices in total Hbs increases the sensitivity of the technique to the detection of only 0.5% HbA in the total Hb mixture, the lower limit of conventional IEF being only 2.5% HbA. Of 15 fetuses from couples at risk analyzed in the region of Ozieri, three were found to be homozygous beta-thalassemic, eight heterozygous, and four normal with no false-positives or -negatives.

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Application of cellulose acetate electrophoresis to globin chain separation for antenatal diagnosis of beta thalassemia

Cited by 11 publications

References 6 publications

Rapid and Sensitive Assessment of Globin Chains for Gene and Cell Therapy of Hemoglobinopathies

Rapid and Sensitive Assessment of Globin Chains for Gene and Cell Therapy of Hemoglobinopathies

Simplified hemoglobin chain detection by capillary electrophoresis

Antenatal diagnosis of β‐thalassemia by isoelectric focusing in immobilized pH gradients

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