Autofluorescence Measurements Can be Used to Differentiate Normal, Activated or Malignant Lymphocyte Populations

Abstract

Purpose:: The diagnosis and management of patients with ocular disorders are largely dependent upon subjective clinical examinations. The goal of this study was to investigate whether emission of autofluorescence from normal, activated or malignant lymphocytes could facilitate clinical diagnosis of ocular diseases.

Methods:: Peripheral blood mononuclear cells (PBMCs) were isolated from normal human donor buffy coat blood products. PBMCs with or without stimulation (PMA and Ionomycin treatment) were cultured at 37 degrees at a concentration of 2 x 10⁶ / mL. At 0, 1, and 4 days after culturing, 0.6x10⁶ cells from each cell group were cyto-spun onto a slide and imaged with a confocal microscope. Six excitation wavelengths were used for imaging: 351, 364, 458, 476, 488, and 514nm. For each excitation condition, the autofluorescence intensity over a range of emission wavelengths was recorded. Alternatively, T-cells were purified from PBMCs and cultured with or without anti-CD3 and anti-CD28 stimulation. A B-lymphoma cell line (CA46) was compared to the purified T-cells. Five experimental groups were compared: unstimulated T-cell, stimulated T-cell, CA46 cell, stimulated T-cell mixed with CA46 cell at a ratio of 1:4, or mixed at a ratio of 4:1. The cells were cyto-spun onto slides 0, 1, and 3 days after culturing and imaged as described above.

Results:: After 24 hours of culturing, the stimulated PBMCs showed marginally increased autofluorescence intensity spectra with all six excitation wavelengths, as compared to the unstimulated PBMCs. Four days after culture, the intensity differences were further enhanced, especially when excited with 364, 488, and 514nm light.Pure T- and B-lymphoma populations were clearly distinguishable based on autofluorescence intensity spectra. B-lymphoma cells were the least fluorescent when excited with 351nm light, but were most fluorescent when excited with visible wavelengths including 476 and 488nm. Mixed populations of T- and B-lymphoma cells had emission intensities that fell in-between those of the pure populations.

Conclusions:: Normal, activated and malignant lymphocyte populations can be distinguished based on their autofluorescent properties in vitro. Future work with in vivo models may prove useful in facilitating the diagnosis of uveitis and other ocular diseases.

Keywords: imaging/image analysis: non-clinical • optical properties