Application of dynamic capillary isoelectric focusing to the analysis of human hemoglobin variants

Molteni, Sarah; Frischknecht, Hannes; Thormann, Wolfgang

doi:10.1002/elps.1150150105

Cited by 92 publications

(34 citation statements)

References 21 publications

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“…For separation of proteins with close pI values, the mixture of several ampholytes from different manufacturers reinforces the pI range of interest, giving rise to better resolution than a single ampholyte [12,14,26,27]. Thus, nature and composition of different pI-range ampholytes in the sample mixture was studied in this work.…”

Section: Effect Of Type Concentration and Ratio Of Ampholytesmentioning

confidence: 98%

CIEF with hydrodynamic and chemical mobilization for the separation of forms of α‐1‐acid glycoprotein

Lacunza¹,

Diez-Masa²,

Frutos³

2007

Electrophoresis

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Abstractα‐1‐Acid glycoprotein (AGP) is a protein that exists in different forms, which is due to variations in the amino acid sequence and/or in the glycosidic part of the protein. These differences confer to these forms, among other characteristics, diverse pIs. Changes in these forms of AGP have been correlated to modifications of the pathophysiological conditions of the individuals. One of the analytical techniques employed for their study has been IEF performed in slab gels. CIEF method with hydrodynamic and chemical mobilization, involving an isotachophoretic process, is developed in this work to separate up to 12 bands of forms of standard AGP, which is proposed as a more reproducible, quantitative, less sample‐consuming, and more automated one than conventional IEF. The challenge of this work has been the development of a CIEF method for the separation of bands of a very acidic protein (pI range: 1.8–3.8) in a capillary. Intraday RSD values ≤ 1.7% have been achieved for the relative migration time of the AGP bands to that of an internal standard. For intraday area precision, RSD (%) in the range of 2.70–22.71% for AGP zones accounting for more than 10% of total area of AGP sample has been obtained. As a proof of the potential of the methodology proposed, an AGP sample purified from a pool of sera of patients suffering from ovary cancer is analyzed by CIEF.

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Section: Effect Of Type Concentration and Ratio Of Ampholytesmentioning

confidence: 98%

CIEF with hydrodynamic and chemical mobilization for the separation of forms of α‐1‐acid glycoprotein

Lacunza¹,

Diez-Masa²,

Frutos³

2007

Electrophoresis

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“…However, the series of peaks migrating after Hb A (17-18 min) have not been identified, although the authors suggest that they 'probably represent glycated forms of Hb A, acetylated Hb F and degradation products'. Also Molteni et al [20] have recently investigated the separation of a number of Hb variants (D, S, E), of minor components (Alc and A,) and of umbilical cord blood in newborns. In the latter case, although their patterns prove that there is sample separation between Hb A and Hb F, there is no hint to where Hb Fa, would band in their peak pattern.…”

Section: Introductionmentioning

confidence: 99%

Screening of umbilical cord blood hemoglobins by isoelectric focusing in capillaries

1995

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Separation and quantitation of the three main hemoglobin components of umbilical cord blood (fetal, acetylated fetal and adult hemoglobins; Hb F, Fac, A) by capillary isoelectric focusing (IEF) in a pH 6-8 gradient is reported. Even in coated capillaries (with covalently bound chains of linear acrylamido derivatives, notably N-acryloylaminoethoxyethanol), no base line separation is obtained between Hb F and A, although this is routinely achieved in gel slab IEF. However, when the carrier ampholyte buffers were added to 3% short-chain liquid linear polyacrylamide, base line resolution and stabilization of peak transit times were obtained. This suggests that even in the best coating procedures, patches of the inner capillary surface could still be naked, so that the static coating is complemented by a dynamic coating on the unoccupied sites. An additional improvement in separation occurs if the above mixture, comprising 5% carrier ampholytes in the pH 6-8 range and 3% soluble polyacrylamide, is made to contain 50 mM beta-alanine, a "separator" known to flatten the pH gradient around pH 7. In the normal newborns analyzed (n = 30), the following average values were obtained: Hb F, 70.1% (range 65-75%); Hb A, 20.2% (range 15-25%); and HbFac, 9.5% (range 7-11%).

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“…5,6,13 The ability to assess Hb variants using such a small sample size makes this method ideal for use in both premature and term infants. As shown in Figure 3, capillary isoelectric focusing can identify very small amounts of abnormal Hb variants.…”

Section: Discussionmentioning

confidence: 99%

Effect of Transfusion on Hemoglobin Variants in Preterm Infants

Barkemeyer

Hempe

2000

J Perinatol

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OBJECTIVE:To determine the effect of transfusion on hemoglobin (Hb) variants in very low birth weight infants and to correlate these changes with parameters measured in routine complete blood counts. METHODS:Hb variants were measured by capillary isoelectric focusing on 126 specimens from 25 very low birth weight infants during their hospital course. These results were compared with transfusion frequency, mean corpuscular volume (MCV), and red cell distribution width. RESULTS:Mean initial Hb F level before transfusion was 87.1 Ϯ 5.1%, and a single 15 ml/kg packed red blood cell transfusion decreased the mean Hb F level to 54.0 Ϯ 4.7%. With frequent transfusions in the first month of life, there was progressive decline in Hb F content such that Hb F made up Ͻ15% of the total Hb after five transfusions. There was a linear correlation between Hb F content and MCV, with a correlation coefficient (r) of 0.77 and a p value of Ͻ0.0001. In most instances, when the MCV fell below 100 fl, the Hb F content was Ͻ50%; when the MCV fell below 95 fl, the Hb F content was Ͻ25%. There was a nonlinear correlation between Hb F content and red cell distribution width. CONCLUSION:Transfusion in very low birth weight infants results in a rapid transition from Hb F to Hb A predominance. This transition is marked by a reduction in MCV that allows for prediction of the Hb F content.

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Application of dynamic capillary isoelectric focusing to the analysis of human hemoglobin variants

Cited by 92 publications

References 21 publications

CIEF with hydrodynamic and chemical mobilization for the separation of forms of α‐1‐acid glycoprotein

CIEF with hydrodynamic and chemical mobilization for the separation of forms of α‐1‐acid glycoprotein

Screening of umbilical cord blood hemoglobins by isoelectric focusing in capillaries

Effect of Transfusion on Hemoglobin Variants in Preterm Infants

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