Detection of methemoglobin in whole blood based on confocal micro-Raman spectroscopy and multivariate statistical techniques.

Raman spectroscopy has been shown to have the potential for revealing oxygenated and spin ability of hemoglobin. In this study, confocal micro-Raman spectroscopy is developed to monitor the effect of sodium nitrite on oxyhemoglobin (HbO2 ) in whole blood. We observe that the band at 1,638 cm(-1) which is sensitive to the oxidation state decreases dramatically, while the 1,586 cm(-1) (low-spin state band) reduces both in methemoglobin (MetHb) and poisoning blood. Our results show that adding in sodium nitrite lead to the transition from HbO2 (Fe(2+) ) to MetHb (Fe(3+) ) in whole blood, and the iron atom converts from the low spin state to the high spin state with a delocalization from porphyrin plane. Moreover, multivariate statistical techniques, including principal components analysis (PCA) and linear discriminant analysis (LDA) are employed to develop effective diagnostic algorithms for classification of spectra between pure blood and poisoning blood. The diagnostic algorithms based on PCA-LDA yield a diagnostic sensitivity of 100% and specificity of 100% for separating poisoning blood from normal blood. Receiver operating characteristic (ROC) curve further confirms the effectiveness of the diagnostic algorithm based on PCA-LDA technique. The results from this study demonstrate that Raman spectroscopy combined with PCA-LDA algorithms has tremendous potential for the non-invasive detection of nitrite poisoning blood.