Rapid determination of human globin chains using reversed-phase high-performance liquid chromatography

Wan, Jun-Hui; Tian, Pei-Ling; Luo, Weihao; Wu, Bingyi; Xiong, Fu; Zhou, Wanjun; Wei, Xiangcai; Xu, Xiang

doi:10.1016/j.jchromb.2012.05.041

Cited by 12 publications

(8 citation statements)

References 29 publications

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“…19,20 The ζ globin chain level was determined using a previously described method. 21 The expression of cluster of differentiation (CD)44 in mature erythrocytes was assessed by flow cytometry.…”

Section: Hematological and Biochemical Testsmentioning

confidence: 99%

Compound heterozygosity for KLF1 mutations is associated with microcytic hypochromic anemia and increased fetal hemoglobin

et al. 2015

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Krüppel-like factor 1 (KLF1) regulates erythroid lineage commitment, globin switching, and the terminal maturation of red blood cells. Variants in human KLF1 have been identified as an important causative factor in a wide spectrum of phenotypes. This study investigated two unrelated male children in China who had refractory anemia associated with poikilocythemia. These were accompanied by an upregulation of biochemical markers of hemolysis, along with abnormal hemoglobin (Hb) level and elevated reticulocyte counts. Next-generation sequencing revealed that the patients were compound heterozygotes for a KLF1 frameshift mutation c.525_526insCGGCGCC (p.(Gly176ArgfsTer179)) and one of two missense variants, c.892 G4C (p. (Ala298Pro)) and c.1012C4T (p.(Pro338Ser)). The subjects had microcytic hypochromic anemia, and their healthy parents had single mutation. The two missense mutations affected a highly conserved codon in the zinc finger DNA-binding domain of KLF1, but the protein stability was unaffected in K-562 cells. A KLF1-targeted promoter-reporter assay showed that the two mutations reduce the expression of the HBB, BCL11A, and CD44 genes involved in erythropoiesis, with consequent dyserythropoiesis and an α/non-α chain imbalance. A systematic analysis was performed of the phenotypes associated with the KLF1 mutations in the two families, and the clinical characteristics and differential diagnoses of the disease are presented. This is the first report of an autosomal recessive anemia presenting with microcytic hypochromia, abnormal Hb profile, and other distinctive erythrocyte phenotypes, and provides insight into the multiple roles of KLF1 during erythropoiesis.

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Section: Hematological and Biochemical Testsmentioning

confidence: 99%

Compound heterozygosity for KLF1 mutations is associated with microcytic hypochromic anemia and increased fetal hemoglobin

et al. 2015

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“…Toward lowered backpressure and greatest peak capacity and thus possibly reduced separation times, an HPLC column with core-shell technology was chosen. 51 The specific column, a Phenomenex Aeris Widepore XP-C18, was selected as a replacement of a previous fully porous C18 column, 38 , 39 and has certified stability at pH 1.5–9.0, at backpressures up to 600 bar (60 MPa) and at temperatures up to 90°C. To minimize background noise in the analysis of rare samples at far-UV absorbance measurements, the study additionally drew on acetonitrile supplemented with 0.1% TFA without inclusion of other organic solvents as the organic mobile phase.…”

Section: Resultsmentioning

confidence: 99%

“…In line with routine procedures for RP-HPLC analysis of thalassemic samples, 38 , 39 , 50 blood samples were lysed in HPLC-quality distilled water (H 2 O; 4 μL of PB in 996 μL of H 2 O), vortexing and incubating at room temperature for 10 min. For cell culture samples, the sample material was centrifuged at 2,000 RCF for 10 min, re-suspended in 15 μL of H 2 O/300,000 cells, and twice frozen at −80°C and thawed.…”

Section: Methodsmentioning

confidence: 99%

“…The most significant speed improvement in RP-HPLC application to hemoglobinopathies has come about by a change of column materials (C18 instead of C4 resin) in two studies, but without data for cell culture or animal samples. 38 , 39 Moreover, both advanced protocols employed high-pressure gradient systems at speeds of 3.5 mL/min 38 or 2.0 mL/min, 39 at which the low-pressure gradient systems commonly used in research settings cannot achieve satisfactory mixing of the mobile phases used. In both cases, change of column material reduced peak widths and thus reduced run times down to 16 min, but both reports utilized liberal (0.48 μL of blood) 39 to apparently excessive (hemoglobin equivalent to 1 mL of blood) 38 sample material per injection.…”

Section: Introductionmentioning

confidence: 99%

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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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“…Nevertheless, although it is widely used for the detection, quantification and purification of many proteins from biological materials, its clinical application for hemoglobins is still limited due to the long analytical time, complex sample preparation and resolution problems. 40,41 Recently, studies have had success in optimizing the method, 40,41 but in many laboratories the technique is still employed exclusively for research.…”

Section: Introductionmentioning

confidence: 99%

Hemoglobin D-Punjab: origin, distribution and laboratory diagnosis

Torres

Okumura

Silva

et al. 2015

Revista Brasileira de Hematologia e Hemoterapia

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This review discusses hemoglobin D-Punjab, also known as hemoglobin D-Los Angeles, one of the most common hemoglobin variants worldwide. It is derived from a point mutation in the beta-globin gene (HBB: c.364G>C; rs33946267) prevalent in the Punjab region, Northwestern Indian. Hemoglobin D-Punjab can be inherited in heterozygosis with hemoglobin A causing no clinical or hematological alterations, or in homozygosis, the rarest form of inheritance, a condition that is commonly not related to clinical symptomatology. Moreover, this variant can exist in association with other hemoglobinopathies, such as thalassemias; the most noticeable clinical alterations occur when hemoglobin D-Punjab is associated to hemoglobin S. The clinical manifestations of this association can be similar to homozygosis for hemoglobin S. Although hemoglobin D-Punjab is a common variant globally with clinical importance especially in cases of double heterozygosis, hemoglobin S/D-Punjab is still understudied. In Brazil, for example, hemoglobin D-Punjab is the third most common hemoglobin variant. Thus, this paper summarizes information about the origin, geographic distribution, characterization and occurrence of hemoglobin D-Punjab haplotypes to try to improve our knowledge of this variant. Moreover, a list of the main techniques used in its identification is provided emphasizing the importance of complementary molecular analysis for accurate diagnosis.

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Rapid determination of human globin chains using reversed-phase high-performance liquid chromatography

Cited by 12 publications

References 29 publications

Compound heterozygosity for KLF1 mutations is associated with microcytic hypochromic anemia and increased fetal hemoglobin

Compound heterozygosity for KLF1 mutations is associated with microcytic hypochromic anemia and increased fetal hemoglobin

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

Hemoglobin D-Punjab: origin, distribution and laboratory diagnosis

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