Multivariate analysis of mean Raman spectra of erythrocytes for a fast analysis of the biochemical signature of ageing

Ageing of red blood cells (RBC) is a physiological process, fundamental to ensure a proper blood homeostasis that, in vivo, balances the production of new cells and the removal of senescent erythrocytes. A detailed characterization at the cellular level of the progression of the ageing phenomenon can reveal biological, biophysical and biochemical fingerprints for diseases related to misbalances of the cell turnover and for blood pathologies. We applied Principal Components Analysis (PCA) to mean Raman spectra of single cells at different ageing times to rapidly highlight subtle spectral differences associated with conformational and biochemical modifications. Our results demonstrate a two-step ageing process characterized by a first phase in which proteins plays a relevant role, followed by a further cellular evolution driven by alterations in the membrane lipid contribution. Moreover, we used the same approach to directly analyse relevant spectral effects associated to reduction in Haemoglobin oxygenation level and membrane fluidity induced by the ageing. The method is robust and effective, allowing to classify easily the studied cells based on their age and morphology, and consequently to evaluate the biological quality of a blood sample.