Multivariate analysis of mean Raman spectra of erythrocytes for a fast analysis of the biochemical signature of ageing
Graphical abstract
Introduction
In the decades since 1930, when Raman and Landsteiner together collected their Nobel prize for, respectively, the discovery of the expressly-named Raman effect and of human blood groups, many investigations mixing the two findings have been carried out. Consequently, an increasing number of related analytical and diagnostic applications have been proposed and successfully demonstrated. Raman spectroscopy has now proved to be a powerful tool both for the study of whole blood and of its cellular components [[1], [2], [3], [4]]. The first Raman studies on the structure and vibrational properties of isolated Haemoglobin (Hb) dates back to 1970s [5] and were mainly dedicated to the investigation of different signatures of its oxygenated and deoxygenated states. These results have been fundamental in successive analyses of intact and alive erythrocytes [6], which are substantially constituted of Hb and enzymes surrounded by a cellular membrane containing many transmembrane proteins that anchor the supporting cytoskeleton, in a very peculiar architecture called membrane-skeleton [7]. Raman studies on RBCs membrane alone have also been performed by means of artificially isolated “ghosts” and have mainly been devoted to investigate their composition (lipid and protein contribution and conformation) and their fluidity properties [8]. All the information acquired on oxy/deoxy states of Hb and on membrane flexibility proved important to study the cell ageing and to deepen the knowledge on degradation and damaging processes of erythrocytes [9]. More in general, the mechanical, structural and biochemical modifications at the basis of the ageing process of RBCs are important in the study of specific pathologies [10,11] and to analyse the effects of blood storage for transfusion [12,13].
Very recently, many important papers have been published demonstrating the potentiality of Raman spectroscopy as a non-invasive tool for stored red blood cell analysis focusing on mean effects of haemolysis [14,15], variation of Hb oxygenation [16,17] and age-related changes in supernatant composition, with specific reference to lactate accumulation [18]. In fact, these studies have been performed on dried drops of supernatant [14,18], dry-fixed smears [16] and also directly through sealed transfusion bags, in order to preserve storage sterility and to classify blood quality and its correlation with the time of storage in blood bank [15,17].
In this regard, the ability of Raman microscopy to perform rapid and label-free characterization and spatial mapping of samples appears particularly attractive and already allowed to gain valuable insight into the differences between “old” and “young” erythrocytes at the single cell level [19,20]. As an example, it was demonstrated that in aged RBC, Hb tends to aggregate, partially loses its oxygenation ability and speed and is not homogeneously distributed across the cell, but it is mainly bound to the plasma membrane [19]. This uneven distribution, possibly contributes to reduce the cell's elasticity and deformability. It was also suggested that this behaviour of the Hb, triggered by the ageing process and correlated with the increase of cell density, can produce structural changes and higher exposition of tyrosine residue [13], leading to an increase in the globin-to-heme separation and resulting in a final breakdown of the globin [12]. The ageing pattern also produces serious alterations at the level of the plasma membrane. The total content of sialic acid of old RBCs is expected to be lower than in young cells [[21], [22], [23]], while alterations in the membrane structure and function have been observed and hypothesized to be preceded by effects on the protein components [12]. However, to date, the correlation between modifications of cell properties and ageing is not completely clear and most of the reported Raman results are based on the identification of small intensity differences between selected bands.
Here, we applied Raman imaging to collect a large number of mean spectra of single dried cells at different ageing times. The mean cell spectra were, then, collectively analysed by means of Principal Component Analysis (PCA). This approach allowed to highlight rapidly the main spectral differences associated with conformational and biochemical modifications induced by the ageing process. In particular, we identified a time threshold between two different phases of the process: a first period in which proteins play a relevant role, followed by a second phase driven by alterations in the membrane lipid contribution. Finally, we used the same approach to analyse the relevant spectral effects associated with the reduction in cell oxygenation level and membrane fluidity induced by the ageing. The method and the protocols here defined, which have been applied to RBCs from two different donors to underline their robustness and effectiveness, were studied using the Quadratic Discriminant Analysis (QDA). This provided a quantitative classification of the studied cells on the basis of their age and morphology, which can be used for a rapid evaluation of the quality of blood samples.
Section snippets
Sample preparations
The blood samples have been obtained, for the purpose of scientific research only, from healthy donors after written informed consensus, as required by Italian “National Bioethics Committee”. All the preparation methods and sample manipulations have been performed in accordance to the relevant guidelines and regulations approved by the “Research Ethics and Bioethics Committee” of the Italian CNR.
Blood samples were obtained using venipuncture into Vacutainers (Becton-Dickinson, Franklin Lakes,
Results and discussion
The Raman microscope allowed to collect several optical images and spectral maps on the smears prepared, in the presence of plasma, at different ageing times. Fig. 1 presents an optical image and the correlated spectral map of four different cell morphologies (respectively, from top left to bottom down in the image, spiculed, biconcave, flat and crenated). During the ageing pathway the abundance of these four morphologic phenotypes changes, with each form that is predominant at a different time
Conclusions
We applied Principal Components Analysis (PCA) to mean vibrational spectra of single erythrocytes obtained by Raman mapping to rapidly evidence specific spectral differences associated to the progression of cell ageing. This is a fundamental process for the erythrocytes that, both in vivo and in vitro, is accompanied by the development of severe morphological alterations that are driven by, or associated to, chemical modifications of the cell architecture.
In this framework, our results
Author contribution
E. L. realized imaging experiments, performed multivariate analysis of spectroscopic data and contributed to data analysis and to writing the paper (Investigation, Formal Analysis, Writing); S. D., G. L. and M. G. prepared samples and contributed to data analysis and to writing the paper (Resources, Methodology, Formal Analysis, Writing); V. M. designed research, performed experiments, contributed to data analysis and to writing the paper (Conceptualization, Validation, Investigation, Formal
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
References (42)
- et al.
Raman spectroscopy of blood samples for forensic applications
Forensic Sci. Int.
(2011) - et al.
Imaging human erythrocyte spectrin with atomic force microscopy
Micron
(1994) - et al.
Raman spectroscopic analysis of Dutch Belt rabbit erythrocyte ghosts
Chem. Phys. Lipids
(1976) - et al.
Achievements in resonance Raman spectroscopy review of a technique with a distinct analytical chemistry potential
Anal. Chim. Acta
(2008) - et al.
Structure and function in native and pathological erythrocytes: a quantitative view from the nanoscale
Micron
(2012) - et al.
Human erythrocytes analyzed by generalized 2D Raman correlation spectroscopy
J. Mol. Struct.
(2014) - et al.
Rheologic properties of senescent erythrocytes: loss of surface area and volume with red blood cell age
Blood
(1992) - et al.
The how, when, and why of the aging signals appearing on the human erythrocyte membrane: an atomic force microscopy study of surface roughness
Nanomed. Nanotechnol. Biol. Med.
(2010) - et al.
Probing oxidative stress in single erythrocytes with Raman Tweezers
J. Photochem. Photobiol. B Biol.
(2010) - et al.
Micro-Raman spectroscopy study of the effect of Mid-Ultraviolet radiation on erythrocyte membrane
J. Photochem. Photobiol. B Biol.
(2012)
Red cell age effects on metabolism and oxygen affinity in humans
Respir. Physiol.
Raman spectroscopy of blood and blood components
Appl. Spectrosc.
Multicomponent blood analysis by near-infrared Raman spectroscopy
Appl. Optic.
Mechanochemistry of single red blood cells monitored using Raman tweezers
Biomed. Optic Express
Circulating cardiac troponin I in severe congestive heart failure
Circulation
Raman spectroscopy--a prospective tool in the life sciences
ChemPhysChem : a European journal of chemical physics and physical chemistry
Mechanical response of human red blood cells in health and disease: some structue-property-function relationships
J. Metr. Res.
Human red blood cell aging: correlative changes in surface charge and cell properties
J. Cell Mol. Med.
Characterization of storage-induced red blood cell hemolysis using Raman spectroscopy
Lab. Med.
Non-invasive analysis of stored red blood cells using diffuse resonance Raman spectroscopy
Analyst
Using Raman spectroscopy to assess hemoglobin oxygenation in red blood cell concentrate: an objective proxy for morphological index to gauge the quality of stored blood?
Analyst
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