Raman spectroscopic differentiation of activated versus non-activated T lymphocytes: an in vitro study of an acute allograft rejection model.

Acute rejection (AR) remains a significant complication in renal transplant patients. Using serum creatinine for AR screening has proven problematic, and thus a noninvasive, highly sensitive and specific test is needed. T cells from human peripheral blood were analyzed using Raman spectroscopy. Fifty-one Mixed Lymphocyte Culture (MLC) activated T cells (ATC), 28 Mitomycin C inactivated T cells (ITC), and 35 resting T cells (RTC), were studied utilizing 785 and 514.5 nm wavelengths. Statistical analysis following subtraction of fluorescence used Student's t test to quantify peak ratio differences and discriminant function analysis (DFA), with three distinct sectors assigned for grouping purposes: Sector I, ITC; Sector II, ATC; Sector III, RTC. Differences between ATC and non-activated T cells (ITC and RTC) were found at 1182 and 1195 cm-1 peak positions for both wavelengths. Significant differences in peak ratios for 785 and 514.5 nm wavelengths existed between ATC and RTC (p=0.001 and p=0.006, respectively) and ATC and ITC (p=0.001 and p=0.001, respectively), with a trend in differences observed between ITC and RTC (p=0.07 and p=0.08, respectively). Analysis of the DFA-derived sector distribution for the 785 and 514.5 nm wavelengths revealed a sensitivity of 95.7% and 89.3%, respectively, and a specificity of 100% and 93.8%, respectively. This data suggests that Raman spectroscopy can detect significant differences between activated and nonactivated T cells based upon cell-surface receptor expression, thereby establishing unique signatures that can aid in the development of a noninvasive AR screening tool with high sensitivity and specificity.