Reliable cell preparation protocol for Raman imaging to effectively differentiate normal leukocytes and leukemic blasts
Anna Maria Nowakowska1, Aleksandra Borek-Dorosz1, Patrycja Leszczenko1, Adriana Adamczyk1, Anna Pieczara2, Justyna Jakubowska3, Agata Pastorczak3, Kinga Ostrowska3, Katarzyna Maria Marzec4*, Katarzyna Majzner1,2*
1Faculty of Chemistry, Jagiellonian University, Kraków, Poland
2Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Bobrzynskiego 14, 30‑348 Krakow, Poland
3Department of Pediatrics, Oncology and Hematology, Medical University of Łódź, Łódź, Poland
4Lukasiewicz Research Network—Krakow Institute of Technology, Krakow, Poland
Leukemias are a remarkably diverse group of malignancies originating from abnormal progenitor cells in the bone marrow. Leukemia subtypes are classified according to the cell type that has undergone neoplastic transformation using demanding and time-consuming methods. Alternative is Raman imaging that can be used both for living and fixed cells. However, considering the diversity of leukemic cell types and normal leukocytes, and the availability of different sample preparation protocols, the main objective of this work was to verify them for leukemia and normal blood cell samples for Raman imaging.
The effect of glutaraldehyde (GA) fixation in a concentration gradient (0.1%, 0.5%, and 2.5% GA) on the molecular structure of T-cell acute lymphoblastic leukemia (T-ALL) and peripheral blood mononuclear cells (PBMCs) was verified. Changes in the secondary structure of proteins within cells were indicated as the main effect of fixation, as shown by an increase in band intensity at 1041 cm-1, characteristic for in-plane δ(C-H) deformation in phenylalanine (Phe).
Different sensitivity of mononuclear and leukemic cells to fixation was observed. While the 0.1% concentration of GA was too low to preserve the cell structure for an extended period of time, a GA concentration of 0.5% seemed optimal for both normal and malignant cells. Chemical changes in PBMCs samples stored for 11 days were also investigated, which manifested in numerous modifications in the secondary structure of proteins and the content of nucleic acids. The impact of cell preculturing for 72 h after unbanking was verified, and there was no significant effect on the molecular structure of cells fixed with 0.5% GA. In summary, the developed protocol for the preparation of samples for Raman imaging allows for the effective differentiation of fixed normal leukocytes from malignant T lymphoblasts.
This work was supported by “Label-free and rapid optical imaging, detection and sorting of leukemia cells” project, which is carried out within the Team-Net programme (POIR.04.04.00-00-16ED/18-00) of the Foundation for Polish Science co-financed by the European Union under the European Regional Development Fund.