Rapid determination of human globin chains using reversed-phase high-performance liquid chromatography.
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 × 4.6 mm) with UV detection at 280 nm. Under the conditions of varying pH or the HPLC gradient, the globin chains (pre-β, β, δ, α, (G)γ and (A)γ) 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 β thalassemia) were found in the area ratio of α/pre-β+β applying the rapid elution procedure, while P≥0.05 was obtained between the normal and α thalassemia silent/trait group. Based on the ANOVA results, receiver operating characteristic (ROC) curve analysis of the δ/β and α/pre-β+β area ratios showed a sensitivity of 100.0%, and a specificity of 100.0% for indicating β 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 δ/β for β thalassemia carriers and 0.626 of α/pre-β+β 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.
Department of Medical Genetics, School of Basic Medical Sciences, Southern Medical University, Guangzhou, Guangdong 510515, PR China.
Jun-Hui
JH
Wan
Pei-Ling
PL
Tian
Wei-Hao
WH
Luo
Bing-Yi
BY
Wu
Fu
F
Xiong
Wan-Jun
WJ
Zhou
Xiang-Cai
XC
Wei
Xiang-Min
XM
Xu
D000328:Adult; D000704:Analysis of Variance; D016022:Case-Control Studies; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D006418:Heme / Q000737:chemistry; D055554:Hemoglobin Subunits / Q000032:analysis* / Q000737:chemistry / Q000145:classification; D006455:Hemoglobins, Abnormal / Q000032:analysis* / Q000737:chemistry / Q000145:classification; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007231:Infant, Newborn; D012372:ROC Curve; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013789:Thalassemia / Q000097:blood; D017085:alpha-Thalassemia / Q000097:blood
D055554:Hemoglobin Subunits; D006455:Hemoglobins, Abnormal; D006418:Heme