A preliminary evaluation of attenuated total reflection Fourier transform infrared spectroscopy for the hematological analysis of thalassemias.

OBJECTIVES: The effectiveness of attenuated total reflection Fourier transform infrared spectroscopy for the hematological analysis of thalassemias was evaluated. DESIGN AND METHODS: The correlations of hemoglobin, mean corpuscular volume and mean corpuscular hemoglobin between routine method and attenuated total reflection Fourier transform infrared spectroscopy were analyzed using linear regression analysis. Appropriate cut-off values of predicted mean corpuscular volume and predicted mean corpuscular hemoglobin in screening of thalassemias were derived from the receiver operator characteristic curve conducted on 103 subjects. RESULTS: Obvious positive correlations of hemoglobin (beta=0.876, R(2)=0.791, P<0.001), mean corpuscular volume (beta=0.656, R(2)=0.516, P<0.001) and mean corpuscular hemoglobin (beta=0.674, R(2)=0.583, P<0.001) were observed between routine method and attenuated total reflection Fourier transform infrared spectroscopy. Based on the receiver operator characteristic curve analysis, the best cut off value of predicted mean corpuscular volume for the phenotype-positive subjects was found to be 79.9 fl with a sensitivity of 100.0% and a specificity of 97.8%, and the proposed cut off value of predicted mean corpuscular hemoglobin was 27.3 pg with a sensitivity of 100.0% and a specificity of 96.8%. The area under curve was 0.996 for predicted mean corpuscular volume and 0.992 for predicted mean corpuscular hemoglobin, respectively. CONCLUSIONS: The established method could be an additional potentially promising tool for the preliminary screening of thalassemias in population prevention and control program. The main advantage of this method is no unwanted chemical regents compared with conventional method. Strategy for the development of this method could be of use for the other important parameters of thalassemias.

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-ß, ß, 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 ß thalassemia) were found in the area ratio of α/pre-ß+ß 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/ß and α/pre-ß+ß area ratios showed a sensitivity of 100.0%, and a specificity of 100.0% for indicating ß 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/ß for ß thalassemia carriers and 0.626 of α/pre-ß+ß 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.