Details

Autor(en) / Beteiligte

• Maria Nowakowska, Anna
• Borek-Dorosz, Aleksandra
• Leszczenko, Patrycja
• Adamczyk, Adriana
• Pieczara, Anna
• Jakubowska, Justyna
• Pastorczak, Agata
• Ostrowska, Kinga
• Marzec, Katarzyna Maria
• Majzner, Katarzyna

Titel

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

Ist Teil von

• Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy, 2023-05, Vol.292, p.122408, Article 122408

Ort / Verlag

England: Elsevier B.V

Erscheinungsjahr

2023

Quelle

MEDLINE

Beschreibungen/Notizen

• [Display omitted] •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. 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(CH) 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.