Volume 54, Number 3, 2000 APPLIED SPECTROSCOPY 353 0003-7028 / 00 / 5403-0353$2.00 / 0 q 2000 Society for Applied Spectroscopy Fourier Transform Infrared Spectroscopy as a Method for Monitoring the Molecular Dynamics of Lymphocyte Activation BAYDEN R. WOOD, BRIAN TAIT, and DONALD M C NAUGHTON * Department of Chemistry, Monash University, Wellington Rd., Clayton, Victoria, 3168, Australia (B.R.W., D.M.); and Victorian Transplantation and Immunogenetics Service±Australian Red Cross Blood Services, 2nd Floor Rotary Bone Marrow Research Building, Royal Melbourne Hospital, Grattan Street, Parkville, Victoria, 3052, Australia (B.T.) In this paper we report the application of Fourier transform infra- red (FT-IR) microspectroscopy to monitor the molecular dynamics of lymphocyte activation. Infrared spectra of lymphocytes stimulat- ed with the mitogen phytohaemagglutinin-L show spectral features 15 min after initial stimulation that are not apparent in resting lymphocytes. By analyzing the second-order derivatives of the raw spectra and applying principal components analysis (PCA), we con- clude that the major spectral changes observed in the ®rst hour result from an increase in overall RNA synthesis. Bands character- istic of RNA at 1244, 1080, 1050, 970, 1160, and 1120 cm 21 appear progressively more intense over time in the spectra of activated lymphocytes. The magnitude of these changes increases over time as the cell differentiates into a blast cell. The sensitivity of infrared spectroscopy to RNA moieties and the rapidity of the technique suggest a possible future role for FT-IR spectroscopy in histocom- patibility testing. Index Headings: Fourier transform infrared spectroscopy; Lympho- cyte activation; Phytohaemagglutinin; RNA synthesis; Second-order derivative analysis; Principal components analysis. INTRODUCTION Over the last decade Fourier transform infrared (FT- IR) spectroscopy has been widely applied to the analysis of whole cell preparations, and its potential as a taxo- nomic tool to identify bacteria 1 and as a diagnostic in- dicator for cancer has been the subject of a number of investigations by ourselves and others. 2±9 An advantage infrared spectroscopy has over conventional optical mi- croscopy, in medicine, is the sensitivity of the technique to detect chemical changes that may precede the mor- phological or symptomatic manifestation of disease. 10 The diagnostic capability of FT-IR spectroscopy essen- tially relies on macromolecular differences between dis- eased and nondiseased cells. The infrared spectrum is representative of the total chemical composition of the entire cell population encapsulated by the light path. The spectroscopic changes observed relate to changes in the concentration and conformational orientation of function- al groups associated mainly with proteins, lipids, nucleic acids, and carbohydrates. While a number of FT-IR stud- ies have focused on detecting leukaemic lymphocytes, 7±9 no study has investigated the potential of the technique in histocompatibility testing. In this study we use infrared spectroscopy to monitor the macromolecular changes as- sociated with the lymphocyte activation process. Lymphocytes have a number of properties that enable Received 11 August 1999; accepted 29 October 1999. * Author to whom correspondence should be sent. the study of physiological functions such as division, dif- ferentiation, and molecular recognition. A single lympho- cyte, for instance, is capable of responding to one or more of a limited number of antigens with a similar molecular structure through the antigen speci®city of cell-surface receptors. Antigenic stimulation initiates changes in both the concentration and structure of various macromole- cules and thus is ideally suited to spectroscopic analysis and, in particular, FT-IR analysis. Lymphocyte activation initiated by antigenic stimula- tion is characterized by a complex and intricate series of regulated events in which a resting lymphocyte is stim- ulated to divide and produce progeny. Activation can be induced in vitro by a group of cross-linking agents known as mitogens, which activate T- and B-cells by cross-link- ing surface glycoproteins. Phytohaemagglutinin (PHA), a glycoprotein extracted from the red kidney bean Phas- eolus vulgaris, belongs to a group of compounds known as lectins, which include a large collection of carbohy- drate-binding multimeric compounds derived from plant and animal tissue. 11 PHA binding to N-acetyl- D-galato- samine-containing oligonucleosides initiates a biochemi- cal cascade that characterizes the lymphocyte activation pathway. 11 PHA in combination with the cytokine inter- leukin 2 (IL-2) results in proliferation 72 h after initial stimulation. 12 This cascade involves changes in the mac- romolecular chemistry that can be monitored with FT-IR spectroscopy. In this paper we show that cell stimulation changes detected conventionally as proliferation after 3±4 days can be detected within 15 min by using infrared spec- troscopy. We argue that the spectral changes observed within the ®rst hour following lymphocyte stimulation are the result of RNA synthesis, while changes observed over 48 h are the combined result of RNA and DNA synthesis. The sensitivity of infrared spectroscopy to RNA synthesis indicates a potential role for infrared spectroscopy in histocompatibility testing. EXPERIMENTAL Isolation of Peripheral Blood Mononuclear Cells. Blood was obtained by venipuncture from healthy vol- unteers and placed in glass tubes containing acid citrate dextrose as an anti-coagulant. Peripheral blood mono- nuclear cells (PBMCs) consisting mainly of lymphocytes and monocytes were isolated by the Ficoll-Isopaque (Lymphoprep, Nyegard, Norway) density gradient cen- trifugation technique. 13 The majority of platelets were re-