Human red blood cell behaviour in hydroxyethyl starch: probed by single cell spectroscopy

Abstract

Hydroxyethyl starch (HES) is a commonly used intravenous fluid in hospital settings. The merits and demerits of its application is still a debatable topic. Investigating the interaction of external agents like intravenous fluids with blood cells is of great significance in clinical environments. Micro-Raman spectroscopy combined with an optical tweezers technique has been utilized for conducting systematic investigations of single live red blood cells (RBCs) under the influence of external stress agents. The present work deals with a detailed biophysical study on the response of human live red blood cells in hydroxyethyl starch using optical techniques. Morphological changes in red blood cells were monitored using quantitate phase imaging techniques. Micro-Raman studies suggest that there is a significant reduction in the oxy-haemoglobin level in red blood cells suspended in HES. The spectra recorded by using different probe laser powers has shown that the cells are more vulnerable in HES under the influence of externally induced stress than in blood plasma. In addition, the spectral results support the possibility of heme aggregation and membrane damage for red blood cells in HES under externally induced stress. Principle component analysis performed on the Raman spectra were able to effectively discriminate between red blood cells in HES and in blood plasma. The use of Raman tweezers can be highly beneficial in elucidating biochemical alterations happening in live, human red blood cell.

Fig. 1. Schematic of the indigenously built Raman Tweezers instrument.

Fig. 2. Chemical structure of (a) oxygenated heme and (b) deoxygenated heme.

Fig. 3. Raman spectra for RBCs in control and HES at four different laser powers (a) ∼3 mW (b) ∼5 mW, (c) ∼7 mW and (d) ∼11 mW.

Fig. 4. Bar diagram indicating the oxy-deoxy hemoglobin ratios obtained for RBCs in control and HES at different laser powers for (a) methine deformation region and (b) and (c) spin marker region.

Fig. 5. Bar diagram indicating the intensity variation of the Raman frequency at (a) 674 cm⁻¹ and (b) 565 cm⁻¹.

Fig. 6. Bar diagram indicating the intensity variation of the Raman frequency (a) 752 cm⁻¹ and (b) 999 cm⁻¹.

Fig. 7. PCA plot for Raman spectra for RBCs in control and HES at (a) ∼3 mW (b) ∼5 mW (c) ∼7 mW and (d) ∼11 mW.

Fig. 8. Loading plots for scores of factor 1 (PC1) obtained at different laser powers (3 mW, 5 mW, 7 mW and 11 mW).

Fig. 9. (a, d) Microscopic image (b, e) phase image and (c, f) corresponding 3D view.