Raman-based spectrophenotyping of the most important cells of the immune system

Aleksandra Borek-Dorosz; Anna Maria Nowakowska; Patrycja Leszczenko; Adriana Adamczyk; Anna Pieczara; Justyna Jakubowska; Agata Pastorczak; Kinga Ostrowska; Marta Ząbczyńska; Karol Sowinski; Wieslaw Ignacy Gruszecki; Malgorzata Baranska; Katarzyna Maria Marzec; Katarzyna Majzner

doi:10.1016/j.jare.2021.12.013

Raman-based spectrophenotyping of the most important cells of the immune system

J Adv Res. 2022 Nov:41:191-203. doi: 10.1016/j.jare.2021.12.013. Epub 2022 Jan 4.

Authors

Affiliations

¹ Jagiellonian University, Faculty of Chemistry, Krakow, Poland; Jagiellonian University, Jagiellonian Centre for Experimental Therapeutics (JCET), Krakow, Poland.
² Jagiellonian University, Faculty of Chemistry, Krakow, Poland.
³ Jagiellonian University, Jagiellonian Centre for Experimental Therapeutics (JCET), Krakow, Poland.
⁴ Medical University of Lodz, Department of Pediatrics, Oncology and Hematology, Lodz, Poland.
⁵ Maria Curie-Sklodowska University, Department of Biophysics, Institute of Physics, Lublin, Poland.
⁶ Lukasiewicz Research Network - Krakow Institute of Technology, Krakow, Poland. Electronic address: katarzyna.marzec@kit.lukasiewicz.gov.pl.
⁷ Jagiellonian University, Faculty of Chemistry, Krakow, Poland; Jagiellonian University, Jagiellonian Centre for Experimental Therapeutics (JCET), Krakow, Poland. Electronic address: katarzyna.b.majzner@uj.edu.pl.

Abstract

Introduction: Human peripheral blood mononuclear cells (PBMCs) are a heterogeneous population of cells that includes T and B lymphocytes. The total number of lymphocytes and their percentage in the blood can be a marker for the diagnosis of several human diseases. Currently, cytometric methods are widely used to distinguish subtypes of leukocytes and quantify their number. These techniques use cell immunophenotyping, which is limited by the number of fluorochrome-labeled antibodies that can be applied simultaneously.

Objective: B and T lymphocytes were isolated from peripheral blood obtained from healthy human donors.

Methods: The immunomagnetic negative selection was used for the enrichment of B and T cells fractions, and their purity was assessed by flow cytometry. Isolated cells were fixed with 0.5% glutaraldehyde and measured using confocal Raman imaging. K-means cluster analysis, principal component analysis and partial least squares discriminant methods were applied for the identification of spectroscopic markers to distinguish B and T cells. HPLC was the reference method for identifying carotene in T cells.

Results: Reliable discrimination between T and B lymphocytes based on their spectral profile has been demonstrated using label-free Raman imaging and chemometric analysis. The presence of carotene in T lymphocytes (in addition to the previously reported in plasma) was confirmed and for the first time unequivocally identified as β-carotene. In addition, the molecular features of the lymphocytes nuclei were found to support the discriminant analysis. It has been shown that although the presence of carotenoids in T cells depends on individual donor variability, the reliable differentiation between lymphocytes is possible based on Raman spectra collected from individual cells.

Conclusions: This proves the potential of Raman spectroscopy in clinical diagnostics to automatically differentiate between cells that are an important component of our immune system.

Keywords: B cells; Carotenoids; Confocal Raman imaging; Spectroscopic markers; T cells.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Carotenoids
Discriminant Analysis
Humans
Least-Squares Analysis
Leukocytes, Mononuclear*
Lymphocytes*

Substances

Carotenoids