Discrimination of immune cell activation using Raman micro-spectroscopy in an in-vitro & ex-vivo model

Activation and proliferation of immune cells such as lymphocytes and monocytes are appropriate inflammatory responses to invading pathogens and are key to overcoming an infection. In contrast, uncontrolled and prolonged activation of these cellular signalling pathways can be deleterious to the body and result in the development of autoimmune conditions. The understanding of cellular activatory status therefore plays a significant role in disease diagnosis and progression. Conventional automated approaches such as enzyme linked immunosorbent assays (ELISA) and immune-labelling techniques are time-consuming and expensive, relying on a commercially available and specific antibody to identify cell activation. Developing a label-free method for assessing molecular changes would therefore offer a quick and cost-efficient alternative in biomedical research. Here Raman spectroscopy is presented as an effective spectroscopic method for the identification of activated immune cells using both cell lines and primary cells (including purified monocyte and lymphocyte subgroups and mixed peripheral blood mononuclear cell (PBMC) populations) obtained from healthy donors. All cell lines and primary cells were exposed to different stimulants and cellular responses confirmed by flow cytometry or ELISA. Machine learning models of cell discrimination using Raman spectra were developed and compared to reference flow-cytometry, with spectral discrimination levels comparing favourably with the reference method. Spectral signatures of molecular expression after activation were also extracted with results demonstrating alignment with expected profiles. High performance classification models constructed in these in-vitro and ex-vivo studies enabled identification of the spectroscopic discrimination of immune cell subtypes in their resting and activated state. Further spectral fitting analysis identified a number of potential spectral biomarkers that elucidate the spectral classification.