Infrared spectroscopic identification of beta-thalassemia

The aim of this study was to investigate the potential of infrared (IR) spectroscopy as a fast and reagent-free adjunct tool in the diagnosis and screening of beta-thalassemia.

Blood was obtained from 56 patients with beta-thalassemia major, 1 patient with hemoglobin H disease, and 35 age-matched controls. Hemolysates of blood samples were centrifuged to remove stroma. IR absorption spectra were recorded for duplicate films dried from 5 microL of hemolysate. Differentiation between the two groups of hemoglobin spectra was by two statistical methods: an unsupervised cluster analysis and a supervised linear discriminant analysis (LDA).

The IR spectra revealed changes in the secondary structure of hemoglobin from beta-thalassemia patients compared with that from controls, in particular, a decreased alpha-helix content, an increased content of parallel and antiparallel beta-sheets, and changes in the tyrosine ring absorption band. The hemoglobin from beta-thalassemia patients also showed an increase in the intensity of the IR bands from the cysteine -SH groups. The unsupervised cluster analysis, statistically separating spectra into different groups according to subtle IR spectral differences, allowed separation of control hemoglobin from beta-thalassemia hemoglobin spectra, based mainly on differences in protein secondary structure. The supervised LDA method provided 100% classification accuracy for the training set and 98% accuracy for the validation set in partitioning control and beta-thalassemia samples.

IR spectroscopy holds promise in the clinical diagnosis and screening of beta-thalassemia.