Reliable cell preparation protocol for Raman imaging to effectively differentiate normal leukocytes and leukemic
Anna Maria Nowakowska1, Aleksandra Borek-Dorosz1, Patrycja Leszczenko2
1Jagiellonian University in Kraków, Faculty of Chemistry, Department of Chemical Physics, 2 Gronostajowa St., Krakow, Poland.
Spectrochimica Acta. Part A, Molecular and Biomolecular Spectroscopy
|February 22, 2023
Summary
Raman imaging sample preparation protocols were optimized for leukemia and normal blood cells. A 0.5% glutaraldehyde fixation method effectively differentiates T-cell acute lymphoblastic leukemia from normal leukocytes.
Area of Science:
- Biomedical Optics
- Cellular Biology
- Spectroscopy
Background:
- Leukemia classification relies on complex methods, necessitating advanced diagnostic tools.
- Raman imaging offers a non-destructive method for analyzing both living and fixed cells.
- Standardizing sample preparation for Raman imaging is crucial for accurate leukemia diagnosis.
Purpose of the Study:
- To optimize sample preparation protocols for Raman imaging of leukemia and normal blood cells.
- To evaluate the impact of glutaraldehyde fixation on the molecular structure of T-cell acute lymphoblastic leukemia (T-ALL) and peripheral blood mononuclear cells (PBMCs).
- To establish an effective method for differentiating malignant T lymphoblasts from normal leukocytes using Raman imaging.
Main Methods:
- Investigated the effect of varying glutaraldehyde (GA) concentrations (0.1%, 0.5%, 2.5%) on T-ALL and PBMCs.
- Analyzed molecular structural changes using Raman imaging, focusing on protein secondary structure and phenylalanine.
- Assessed the stability of PBMCs over 11 days and the impact of 72-hour preculturing on fixed cells.
Main Results:
- Glutaraldehyde fixation primarily affects protein secondary structure, indicated by changes in band intensity at 1041 cm⁻¹.
- A 0.5% GA concentration was found to be optimal for preserving both normal and malignant cell structures for Raman imaging.
- PBMCs stored for 11 days showed significant chemical changes, while 72-hour preculturing had no major impact on 0.5% GA-fixed cells.
Conclusions:
- The developed Raman imaging sample preparation protocol effectively differentiates fixed normal leukocytes from malignant T lymphoblasts.
- Optimized glutaraldehyde fixation provides a reliable method for leukemia diagnosis using Raman spectroscopy.
- This approach enhances the potential of Raman imaging as a diagnostic tool in hematological malignancies.
