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.

Acknowledgments

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. This research was partially supported by the Priority Research Area Digiworld under the program Excellence Initiative – Research University at the Jagiellonian University in Kraków and research mini-grant for young and doctoral students of the Faculty of Chemistry as part of the SciMat project, “Development of spectroscopic analytical methodology for leukocyte phenotyping’, PSP N20/MNS/000023. Authors would like to address special gratitude to Ms. Adrianna Wisłocka-Orłowska, Ms. Klaudia Mielnik and Dr. Krzysztof Brzozowski (Jagiellonian University, Faculty of Chemistry, Krakow, Poland) for their kind assistance in data acquisition.