Characterization of Human Platelets by Raman Spectroscopy

Lacroix, Nicolas (University of Ottawa)
Omelon, Sidney (University of Ottawa)
Variola, Fabio (University of Ottawa)

Introduction

Raman spectroscopy is a powerful analytical technique that offers many advantages, in particular a high specificity that allows to precisely determine the physico-chemical makeup of Raman-active materials. Raman spectroscopy is based on the inelastic scattering of photons as a result of the interaction between a monochromatic light and molecular vibrations. Raman spectra contain information about chemical composition, molecular bonding, symmetry and structures, as well as other physical parameters. Recent developments in combining Raman spectroscopy with optical microscopy have provided a new noninvasive strategy to assess the biochemical compositions of individual cells and bacteria. However, this approach has not been applied to the investigation of human platelets. Platelets participate in vital roles in processes such as thrombosis, inflammation and wound healing (Lacci, 2010). However, very little is known about their intracellular chemical composition. To address the challenge, we hypothesized that platelets could be extracted from platelet-rich plasma and investigated by Raman spectroscopy. The objective was to generate Raman spectra from platelets by optimizing sample preparation, laser wavelength and Rama spectra signal collection strategy.

Materials and Methods

Expired platelets obtained from the Canadian Blood Services were frozen at -80 °C, and thawed at 37 °C. Spectroscopic analysis was carried out with a WiTec Alpha 300R confocal Raman microscope. A 488 nm laser was used as excitation source. The laser was focused on dried platelets by using 20X and 50X air-objectives. Single line Raman spectra were taken at maximum laser power, with 10 second acquisition time for 10 accumulations. Raman spectra were compared after normalization. In parallel with these studies, we have exploited complementary chemical assays to support the findings obtained by Raman spectroscopy, using histological and fluorescence microscopy to confirm the Raman spectroscopic identification of platelets components.

Results

The signals generated from suspended platelets in platelet rich plasma were not strong enough to be analyzed by Raman spectroscopy. The platelets were isolated from platelet-rich buffer by gentle centrifugation. Platelet pellets were mounted on polished metal slides, and dried at room temperature to concentrate the platelets. This increased the signal to noise ratio, however, drying also concentrates serum components. To avoid measurement of serum components, platelets were washed with deionized water or buffered saline, and suspended in water, Tris- or citrate-buffered solutions at neutral pH to determine washed platelet stability. Comparison of the platelet Raman spectra as-dried (Fig. 1a), prepared with deionized water (Fig 1b) and shows that at characteristic peak at 1155 cm-1 is lost after 24 hours. The 1155 cm-1 peak correlates with a phosphate species that was confirmed by fluorescence microscopy with DAPI, and histological staining with toluidine blue. This peak remains intact if the platelets are washed in buffered saline instead of water, then suspended in buffer. This 1155 cm-1 peak is obscured by a similar peak generated by Tris buffer (Fig. 2). The Raman spectra of citrate buffer do not overlap with the 1155 cm-1 peak (Fig. 3), so this is a preferable suspension medium.

Discussion and Conclusion

Raman spectroscopy is a powerful technique that offers many advantages, in particular, high spatial and spectral resolution. In this work, we isolated and prepared platelets to exploit this non-destructive spectroscopic technique. Our investigation aims at generating a sample preparation and analysis protocol to advance the current knowledge of the physico-chemical characteristics of platelets by identifying their chemical composition with Raman spectroscopy.

Raman spectra of a) pelleted platelets, and b) platelet pellet, washed and suspended in deionized water

Platelets washed in buffered saline, and resuspended in Tris buffer

Platelets washed in buffered saline, and re-suspended in citrate buffer

Acknowledgements

SO and FV acknowledge funding from NSERC Discovery Grants. FV also acknowledges CFI.

References

Lacci, K.M., and Dardik, A. "Platelet-rich plasma: support for its use in wound healing." The Yale Journal of Biology and Medicine (2010) 83(1), p 1-9.

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