Reliable cell preparation protocol for Raman imaging to effectively differentiate normal leukocytes and leukemic blasts

•Leukemic and normal cells exhibit different sensitivity to glutaraldehyde (GA) fixation.•The stability over time of cells fixed with 0.5% GA is optimal and efficient in comparison to cells fixed with 0.1% GA.•Principal Component Analysis is able to discriminate Raman spectra of cells fixed with different GA concentration, stored over time or precultured prior to fixation.•Spectral analysis revealed alterations in the biochemical composition of mononuclear and leukemic cells due to fixation in the GA concentration gradient.•Raman spectral markers of glutaraldehyde - fixed endothelial cells were identified.