Access Type

Open Access Dissertation

Date of Award


Degree Type


Degree Name



Biomedical Engineering

First Advisor

Gregory Auner







Advisor: Gregory Auner, PhD

Major: Biomedical Engineering

Degree: Doctor of Philosophy

Despite the advances made in the area of kidney transplantation, the disparity between the demand and available donated organs remains a dominant and unresolved issue. Given the paucity of available renal allografts the preservation of existing grafts is vital. One factor that has negatively impacted renal allograft survival is acute rejection (AR). Traditionally, kidney transplant centers have used elevations in serum creatinine as a screening tool for detecting AR. However, with its diagnostic delay, low sensitivity, and low specificity, serum creatinine has proven to be an unreliable and problematic bio-marker. Acute rejection is an activated T lymphocyte driven process that leads to graft dysfunction and possible loss. The activation state of T lymphocytes is determined by the specific cell surface receptor composition present. A technologic tool that could resolve these receptor differences could detect T lymphocyte activation and thus provide a diagnostic modality for AR. Raman spectroscopy (RS), a laser-based technology that is able to characterize substances based on molecular vibrational signatures, represents this modality. Using T lymphocytes isolated from human peripheral blood and clean-catch urine we investigated three aspects of T lymphocyte activation using a modified RS system. First, we explored the sensitivity (the ability to detect activation) of a RS-based system by analyzing mixed lymphocyte reacted (MLR), Mitomycin C inactivated, and resting T lymphocytes at 785nm and 514.5nm wavelengths. Second, the specificity (the ability to distinguish T cells activated by different stimuli) of the system was determined by comparing the signatures of MLR and CD3/CD28-activated T lymphocytes. Third, we analyzed the biomolecular events that conveyed the spectral changes detected by RS. This was carried out by coupling RS analysis of Mitogen-activated T lymphocytes with antigen expression kinetic studies designed to quantify the intensity and timing of cell surface receptor up-regulation. We found that there were significant RS signature differences between the MLR and non-activated (inactivated and resting) T lymphocytes while there was only a trend toward difference seen between the resting and inactivated cellular populations. When analyzing MLR versus CD3/CD28-activated cells, both samples differed from the inactivated and resting groups and demonstrated differences in Raman shifts at multiple foci when compared to one another. Receptor expression kinetics of Mitogen-activated T lymphocytes analyzed at the early and late phases of activation showed differential antibody immuno-fluorescent intensity. This correlated to spectral differences at defined peaks. Moreover, when analyzing all forms of activation (i.e. MLR, CD3/CD28, or Mitogen) there were conserved and reproducible signature changes regardless of the mode of activation which supports the notion that there are receptor and receptor moiety changes that are required in all forms of T lymphocyte activation. This dissertation outlines the use of RS in the resolution and modeling of cell surface receptor differences that define T lymphocyte activation. The accurate detection of T lymphocyte activation within a biomatrix is the foundational step toward the development of a noninvasive tool capable of accurately detecting AR in real-time within the clinical setting.