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

被引：13

作者：

Wan, Jun-Hui ^{[2
]}

Tian, Pei-Ling ^{[1
]}

Luo, Wei-Hao ^{[2
]}

Wu, Bing-Yi ^{[3
]}

Xiong, Fu ^{[2
]}

Zhou, Wan-Jun ^{[2
]}

Wei, Xiang-Cai ^{[1
]}

Xu, Xiang-Min ^{[2
]}

机构：

[1] Family Planning Res Inst Guangdong, Guangzhou 510600, Guangdong, Peoples R China

[2] So Med Univ, Sch Basic Med Sci, Dept Med Genet, Guangzhou 510515, Guangdong, Peoples R China

[3] So Med Univ, Nanfang Hosp, Res Ctr Clin Med, Guangzhou 510515, Guangdong, Peoples R China

来源：

JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES | 2012年 / 901卷

基金：

国家高技术研究发展计划(863计划);

关键词：

Reversed-phase high-performance liquid chromatography; Human globin chain; Thalassemia; Hemoglobin variant; BETA-THALASSEMIA MUTATIONS; HEMOGLOBIN; IDENTIFICATION; SEPARATION; TOOL;

D O I：

10.1016/j.jchromb.2012.05.041

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

Reversed-phase high-performance liquid chromatography (RP-HPLC) of human globin chains is an important tool for detecting thalassemias and hemoglobin variants. The challenges of this method that limit its clinical application are a long analytical time and complex sample preparation. The aim of this study was to establish a simple, rapid and high-resolution RP-HPLC method for the separation of globin chains in human blood. Red blood cells from newborns and adults were diluted in deionized water and injected directly onto a micro-jupiter C18 reversed-phase column (250 mm x 4.6 mm) with UV detection at 280 nm. Under the conditions of varying pH or the HPLC gradient, the globin chains (pre-beta, beta, delta, alpha, (G)gamma and (A)gamma) were denatured and separated from the heme groups in 12 min with a retention time coefficient of variation (CV) ranging from 0.11 to 1.29% and a peak area CV between 0.32% and 4.86%. Significant differences (P < 0.05) among three groups (normal, Hb H and beta thalassemia) were found in the area ratio of alpha/pre-beta+beta applying the rapid elution procedure, while P >= 0.05 was obtained between the normal and a thalassemia silent/trait group. Based on the ANOVA results, receiver operating characteristic (ROC) curve analysis of the delta/beta and alpha/pre-beta+beta area ratios showed a sensitivity of 100.0%, and a specificity of 100.0% for indicating beta thalassemia carriers, and a sensitivity of 96.6% and a specificity of 89.6% for the prediction of hemoglobin H (Hb H) disease. The proposed cut-off was 0.026 of delta/beta for beta thalassemia carriers and 0.626 of alpha/pre-beta+beta for Hb H disease. In addition, abnormal hemoglobin hemoglobin E (Hb E) and Hb Westmead (Hb WS) were successfully identified using this RP-HPLC method. Our experience in developing this RP-HPLC method for the rapid separation of human globin chains could be of use for similar work. (C) 2012 Elsevier B.V. All rights reserved.

引用

页码：53 / 58

页数：6

共 29 条

[1] Prenatal diagnosis in beta-thalassemia: An Indian experience [J].

Agarwal, S ;

Gupta, A ;

Gupta, UR ;

Sarwai, S ;

Phadke, S ;

Agarwal, SS .

FETAL DIAGNOSIS AND THERAPY, 2003, 18 (05) :328-332

[2]

Calvo M.C., 2010, NATURE, V467, P318

[3]

Clarke GM, 2000, CLIN CHEM, V46, P1284

[4] Effective erythropoiesis and HbF reactivation induced by kit ligand in β-thalassemia [J].

Gabbianelli, Marco ;

Morsilli, Ornella ;

Massa, Adriana ;

Pasquini, Luca ;

Cianciulli, Paolo ;

Testa, Ugo ;

Peschle, Cesare .

BLOOD, 2008, 111 (01) :421-429

[5] Haemoglobinopathy analyses in the Netherlands:: a report of an in vitro globin chain biosynthesis survey using a rapid, modified method [J].

Giordano, PC ;

Van Delft, P ;

Batelaan, D ;

Harteveld, CL ;

Bernini, LF .

CLINICAL AND LABORATORY HAEMATOLOGY, 1999, 21 (04) :247-255

[6]

Hatzistavrou T, 2009, NAT METHODS, V6, P659, DOI [10.1038/nmeth.1364, 10.1038/NMETH.1364]

[7] Rapid Identification of Common β-Thalassemia Mutations in the Chinese Population Using Duplex or Triplex Amplicon Genotyping by High-Resolution Melting Analysis [J].

He, Xiang ;

Sheng, Min ;

Xu, Miao ;

Xiong, Can ;

Ren, Zhaorui .

GENETIC TESTING AND MOLECULAR BIOMARKERS, 2010, 14 (06) :851-856

[8] Population genetics of malaria resistance in humans [J].

Hedrick, P. W. .

HEREDITY, 2011, 107 (04) :283-304

[9]

INOUE H, 1989, Z RECHTSMED, V102, P437

[10] Globin chain synthesis is a useful complementary tool in the differential diagnosis of thalassemias [J].

Khatami, Shohreh ;

Dehboneh, Soghra Rouhi ;

Sadeghi, Sedigheh ;

Mirzazadeh, Roghieh ;

Saeedi, Parinaz ;

Bayat, Parastoo ;

Najmabadi, Hossein ;

Zeinali, Sirous ;

Akbari, Mohammad Taghi ;

Ardjmand, Mohammad ;

Amirkhani, Aref .

HEMOGLOBIN, 2007, 31 (03) :333-341

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