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Issue 7, 2015
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Label-free phenotyping of peripheral blood lymphocytes by infrared imaging

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Abstract

It is now widely accepted that the immune microenvironment of tumors and more precisely Tumor Infiltrating Lymphocytes (TIL) play an important role in cancer development and outcome. TILs are considered to be important prognostic and predictive factors based on a growing body of clinical evidence; however, their presence at the tumor site is not currently assessed routinely. FTIR (Fourier transform infrared) imaging has proven it has value in studying a range of tumors, particularly for characterizing tumor cells. Currently, very little is known about the potential for FTIR imaging to characterize TIL. The present proof of concept study investigates the ability of FTIR imaging to identify the principal lymphocyte subpopulations present in human peripheral blood (PB). A negative cell isolation method was employed to select pure, label-free, helper T cells (CD4+), cytotoxic T cells (CD8+) and B cells (CD19+) from six healthy donors PB by Fluorescence Activated Cell Sorting (FACS). Cells were centrifuged onto Barium Fluoride windows and ten infrared images were recorded for each lymphocyte subpopulation from all six donors. After spectral pre-treatment, statistical analyses were performed. Unsupervised Principal Component Analyses (PCA) revealed that in the absence of donor variability, CD4+ T cells, CD8+ T cells and B cells each display distinct IR spectral features. Supervised Partial Least Square Discriminant Analyses (PLS-DA) demonstrated that the differences between the three lymphocyte subpopulations are reflected in their IR spectra, permitting their individual identification even when significant donor variability is present. Our results also show that a distinct spectral signature is associated with antibody binding. To our knowledge this is the first study reporting that FTIR imaging can effectively identify T and B lymphocytes and differentiate helper T cells from cytotoxic T cells. This proof of concept study demonstrates that FTIR imaging is a reliable tool for the identification of lymphocyte subpopulations and has the potential for use in characterizing TIL.

Graphical abstract: Label-free phenotyping of peripheral blood lymphocytes by infrared imaging

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Supplementary files

Article information


Submitted
15 Oct 2014
Accepted
02 Dec 2014
First published
15 Dec 2014

Analyst, 2015,140, 2247-2256
Article type
Paper
Author version available

Label-free phenotyping of peripheral blood lymphocytes by infrared imaging

M. Verdonck, S. Garaud, H. Duvillier, K. Willard-Gallo and E. Goormaghtigh, Analyst, 2015, 140, 2247
DOI: 10.1039/C4AN01855A

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