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.

红血细胞（RBC）的老化是一个生理过程，从根本上确保一个适当的血液内稳态的是，在体内，平衡新细胞的产生和衰老的红细胞的去除。在衰老现象进展的细胞水平上的详细表征可以揭示与细胞更新失衡有关的疾病和血液病理学的生物学，生物物理和生化指纹。我们应用主成分分析（PCA）来表示不同衰老时间下单个细胞的拉曼光谱，以快速突出与构象和生化修饰相关的细微光谱差异。我们的结果证明了两步老化过程，其特征在于蛋白质在其中发挥相关作用的第一阶段，然后是膜脂质贡献的变化驱动的进一步细胞进化。此外，我们使用相同的方法直接分析与衰老引起的血红蛋白氧合水平降低和膜流动性降低相关的光谱效应。该方法是鲁棒且有效的，允许根据其年龄和形态对所研究的细胞进行轻松分类，从而评估血液样本的生物学质量。