Research paper
Raman spectroscopic differentiation of activated versus non-activated T lymphocytes: An in vitro study of an acute allograft rejection model☆

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Abstract

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.

Introduction

Recent advances in immunosuppressive therapy, medical management, and surgical technique have further improved patient and graft outcomes in renal transplantation, thereby expanding the candidate pool to include higher-risk recipients (Foster et al., 2002). Even with use of more marginal deceased-donor organs and pushing the use of live-donor organs to the limit, however, the disparity between supply and demand for organs continues to grow exponentially (Spring, 2007). In the face of a profound organ shortage, it becomes paramount that the longevity of existing grafts be maximized. One condition that negatively impacts this longevity is acute rejection (AR). AR occurs in approximately 15–20% of patients during the first year posttransplant and remains an important factor leading to decreased long-term graft function and survival (Meier-Kriesche et al., 2000); (Hardinger et al., 2001). Reliable screening for AR has proven to be problematic. Serum creatinine, the most commonly used indicator of transplant function (Gaber et al., 1996), is not an accurate reflection of glomerular filtration rate, lacks sensitivity and specificity (Matas et al., 1977), and becomes elevated only after significant histologic damage has occurred (Cosio et al., 1997). Based on the aforementioned observations, a rapid, noninvasive, highly specific and sensitive screening test for AR is warranted.

It has been well established that T lymphocytes are the predominant immune cells involved in the process of AR (Janeway et al., 1999a, Janeway et al., 1999b). The hallmarks of T lymphocyte activation are an increase in cytotoxic specific DNA transcription, immuno-specific proteins (mainly perforin and granzymes) and a characteristic up-regulation of cell surface receptors, notably CD69, CD25, CD71, and CD3(Bernard et al., 1984, Chen et al., 1988, Testi et al., 1988, Testi et al., 1989). We hypothesize that the differentiation of activated and non-activated T lymphocytes, in blood or urine, can be accomplished by using a biomedical tool that analyzes these cell surface receptors.

Raman spectroscopy has been used for years in industry and engineering to characterize and identify materials based upon their energy modes that are unique to that particular substance. Recently, this technique has been extensively applied in the biomedical arena for the detection of a variety of cancers and even pathogens present in the environment (Chan et al., 2006, Xun-Ling et al., 2005, Lyng et al., 2007). Raman spectroscopy utilizes a laser source which interacts with various vibrational and other modes leading to light scattering of the elastic and inelastic nature. The energy spectrum of the inelastic scattered light is directly related to vibrational, rotational and other low energy modes specific to the biochemical composition of the substance under investigation. These energy modes serve as a molecular fingerprint and can be rapidly translated into a unique spectral signature using minute amounts of sample. To our knowledge, this is the first study utilizing Raman spectroscopy to analyze the differences in receptor content found on the cell surface of intact activated and non-activated T lymphocytes for the purpose of developing a methodology to detect AR.

Section snippets

Cell preparation

Following approval from the Wayne State University Human Investigation Committee, mononuclear cells were obtained using freshly drawn (within 3 h of experimentation) sodium-heparinized venous blood collected from healthy individuals and separated according to density via high molecular weight sucrose polymer (ficoll) as described by Boyum (Boyum, 1968). The resultant cells were washed three times using Hank's balanced salt solution (HBSS, Invitrogen, Carlsbad, CA) and suspended in McCoy's

Raman spectroscopy results — red (785 nm) laser

A total of 23 activated, 12 inactivated, and 20 resting T lymphocytes were examined. A representative Raman spectrum of resting T lymphocytes is depicted in Fig. 2 with proposed peak assignments for 1182 cm− 1 and 1195 cm− 1 positions shown in Table 1. There was no observed cellular disruption during measurements, and after comparison of activated and non-activated (resting and inactivated) spectra from 600–1700 cm− 1, qualitative differences were seen at the 1182 cm− 1 and 1195 cm− 1 positions.

Discussion

The purpose of this study was to utilize Raman spectroscopy to establish biomedical-based differences between activated and non-activated T lymphocytes which can serve as the basis of a non-invasive screening modality for AR. Although other noninvasive biomedical technologies to detect AR have been proposed, many require disruption of cellular biology and immunological integrity followed by labor intensive processing (Clarke et al., 2003, Wishart, 2005), while others are limited by the need to

Conclusion

Raman spectroscopy can detect significant differences between activated and non-activated T lymphocytes and thus establish unique signatures for both. These signatures form the basis of a detection system which may facilitate the development of a specific and rapid screening tool for AR of renal transplants.

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    ☆

    This work supported in part by the National Institutes of Health Grant 5R01EB000741-05 and the David Fromm Research Award, Wayne State University Department of Surgery.

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