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Raman Spectroscopy-Based Study of the Discrimination and Oxygen Carrying Capacity of Thalassemia Erythrocytes

Author: ChenXiuLi
Tutor: LiuJunXian；WangGuiWen
School: Guangxi Normal University
Course: Theoretical Physics
Keywords: Raman spectroscopy erythrocyte thalassemia identification oxygen carrying capacity
CLC: Q6-3
Type: Master's thesis
Year: 2009
Downloads: 35
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Abstract

As is well known, blood is composed of blood cells and plasma. Blood cells are suspended in plasma, including erythrocytes, white blood cells and blood platelets. The number of erythrocytes is the largest in the blood cells. And erythrocytes play a very important role in metabolism and gas transportation for human body,which is accomplished by intracellular hemoglobin. The changes of the external environment, such as osmotic pressure, drug treatment, or the pressure of oxygen may induce conformation change of hemoglobin. Peeping into the structure of erythrocyte and hemoglobin would make human understand the oxygen carrying capacity of erythrocyte and the pathology of red blood cells disease, which provided the theoretical basis for diagnosis and treatment. The thalassemias are a group of anemias that result from inherited defects in the production of hemoglobin. The incidence of this disease is very high in south of china. Thalassemias are divided intoα-thalassemia andβ-thalassemia. As a genetic disease, there is no effective way to cure thalassemia at present. To screen thalassemia patients and to avoid children with thalassemia-major born is the only way to control the spread of the disease. However, in conventional detection methods, the cellular information is abtained from a mass of cells or hemoglobin. Besides, those methods necessarily require cumbersome operation as well as heavy workload, and thalassemia treatment cost is relatively expensive, so those methods are not suitable for screening large populations. In addition, the oxygen carrying capacity of thalassemia patient hemoglobin may be different from that of normal control because of the inherent defects of thalassemia hemoglobin. Therefore, developing a rapid, low-cost detection method to identify thalassemia or monitor the dynamic behavior during oxygenanation cycle is very important in clinic.Raman spectroscopy, which offers a noninvasive, nondestructive, fast, and water-insensitive technique, is widely applied in biology and medicine. Raman technique is suitable for biological macromolecules, cells etc, in aqueous solution. This study combined confocal micro-Raman with laser tweezers (In other words, Laser tweezers Raman spectroscopy,LTRS) technique. Optical tweezers could trap a cell. LTRS technique not only eliminated adverse effects caused by common micro-Raman technique because cells must fix on slide, but also obtained a higher signal noise ratio (SNR). It offers unique advantages to the study of single red blood cell, and is expected to diagnosis thalassemia, and study oxygen-carrying function of erythrocyte from normal control or sick. But high-intensity background of spectral signal caused by system noise, fluorescence, etc, would seriously affect the results of statistical analysis based on the intensity of spectra signal. Therefore,spectral data processing effectively is very necessary.In this paper, the LTRS system was used to trap single erythrocyte from normal control,α-thalassemia (HbH, HbH-CS),β-thalassemia major, and to collect the Raman spectra of the trapped cells. A new hybrid algorithm provided by least-squares polynomial fitting was proposed to eliminate the high-intensity background of spectral signal. This paper applied principal component analysis for compressing the Raman spectral vectors and a rapid data classification based on Cluster analysis or back-propagation neural network for predicting different type erythrocyte. In addition, this paper also examined dynamic behavior of oxygen carrying capacity of different erythrocyte, and analyzed individual cellular differences. This paper includes four parts as following:(1) Least-square polynomial fitting was proposed to eliminate the high-intensity background of spetral signal caused by system noise, fluorescence which would seriously interfered the results of statistical analysis .Comparing with the cluster analysis effect of different data processing methods, the results indicated that the discrimination between the erythrocytes obtained from patients and normal person was significantly improved by using the background-removed spectra. The discrimination index betweenα-thalassemia HbH and normal groups was 90.32%, and that betweenα-thalassemia HbH-CS and normal group was 97.6%, which were higher than the results obtained from other methods. In addition, this study also showed that some variations of experimental conditions did not affect the analysis results.(2) Principal component analysis (PCA) algorithm combined with back-propagation neural network predictive model was performed to distinguish abnormal erythrocyte. The PCA results revealed that the difference between normal control andα-thalassemia HbH-CS was significant with the predictive accuracy of BP network as high as 97.90 %. The difference between normal control andβ-thalassemia major,α-thalassemia HbH-CS andβ-thalassemia major were unobvious. The predictive accuracy came next with 90.72 % and 86.28 %, respectively. These results tally closely with the corresponding averaged Raman spectra. Under different experimental condition, the predictive accuracy showed similar results. This pilot study can serve as a useful probe for developing a rapid, simple, reagent-free method for distinguishing of thalassemia erythrocytes. (3) This study combined LTRS technology with gas control device to peep the oxygen carrying capacity of red blood cells. In this work, the effects that laser power on the analysis of oxygenation state was analyzed, and an indicator to distinguish the oxygenated erythrocyte from deoxygenated erythrocyte was chosen. The indicator was utilized to analyze the oxygen carrying capacity of different red blood cell, such as different storage time, different health state. The result showed that, first, strong power led to the bands at 1248, 1371cm-1 as markers for heme aggregation increasing. Second, the ratio I1638/I1547 was the indicator to distinguish oxygenated state from deoxygenated state. Third, with the extension of storage time, erythrocytes were easy to oxygenate, but their deoxidization capacity were not significantly change. Fourth, comparing with normal control, erythrocytes ofα-thalassemia HbH-CS were easy to oxygenate, but difficult to deoxygenate. These findings demonstrate that Laser tweezers Raman spectroscopy could be used to rapidly, sensitively analyze and evaluate the oxygen carrying capacity of erythrocytes.(4) Single erythrocyte fromα-thalassemia HbH-CS patients and normal donors was trapped, and the Raman scattering of trapped cell was collected during ventilation. By comparing the oxygenation cycle of optically trapping single red blood cells and the deoxygenated cells population from the two kinds of specimen, and analyzing the oxygenated and deoxygenated states, it was found that, in the oxygenation cycle, the ratio I1638/I1547 ofα-thalassemia HbH-CS was much higher than that of normal control in deoxygenated state and slightly higher than the normal control in oxygenated state. In the statistic population, 46% of the red blood cells from the normal control reach oxygenated state, butα-thalassemia HbH-CS only 15%, if the base was set by less than the average of I1638/I1547 from normal control cells. The result showed that the red blood cells ofα-thalassemia HbH-CS were easy to oxygenate, but difficult to deoxygenate. The individual difference betweenα-thalassemia HbH-CS erythrocytes was obvious. This study provides the spectral vision on the oxygenation ofα-thalassemia HbH-CS erythrocytes and the laboratorial referrences for the diagnosis and treatment.

Raman Spectroscopy-Based Study of the Discrimination and Oxygen Carrying Capacity of Thalassemia Erythrocytes

Abstract

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